The town of Bottleneck, somewhere in The Wild West, is a typical example of frontier lawlessness, with the customary shootouts and chaos; in the opening scene a horse charges out of a saloon. Said saloon is owned by Kent, a murderous criminal who cheats people out of their ranches in card games and kills them when they complain about it. He is assisted in the card cheating, if not the murder, by "Frenchy", his exotic vaguely European chanteuse (Dietrich). He is also in league with Slade, Bottleneck's corrupt mayor. When Sheriff Keogh comes into the saloon to force Kent to give up a deed he just won by cheating at cards, Kent murders him.
Kent and Slade replace Keogh with Wash, a former deputy who is now a derelict alcoholic, but likes to talk about his glory days riding with Sheriff Tom Destry. Wash takes his job more seriously, though, and sends for his old sheriff's son, Tom Destry Jr. (Stewart). Wash is unpleasantly surprised and Kent is delighted when young Destry shows up and proves to be a mild-mannered fellow who doesn't carry a gun and orders milk at the bar. But Destry is more determined than Kent and Slade guess, and he has a plan.
Destry Rides Again was the first Western James Stewart ever made, and the last until he started making Westerns in the 1950s with Anthony Mann. Anybody who has seen this film and also seen Blazing Saddles knows exactly where Lili von Shtupp comes from. Madeline Kahn wasn't just doing a Marlene Dietrich impersonation; she was doing an impersonation of Dietrich's character in this movie.
- The Alcoholic: Wash is a pretty sad example, passing out in Kent's saloon, shamefacedly admitting to being "the town drunk". But he pulls himself together when he's made sheriff.
- As You Know: Wash does some of this in the first scene to establish that Tom Destry was a famous lawman and Wash was once his deputy.
- Bar Brawl: An unconventional one. Kent and his goons are barricaded inside the saloon. Destry and his posse is outside the saloon, about to take it by storm. A bloody climax seems in the offing. Then the town women, determined to stop the bloodshed, come barreling into the saloon wielding two-by-fours. A wild, chaotic fight scene follows in which the ladies of Bottleneck eventually whack the main bad guys (except for Kent) on the head and take them away.
- Batman Grabs a Gun: The pacifistic Destry grabs a gun when the Sheriff is killed.
- Cat Fight: One of the most famous in cinema, as Frenchy and jealous wife Lily Belle (played by Una Merkel) engage in a slapping, hair-pullling Cat Fight, with Frenchy's legs prominently featured, until Destry pours a bucket of water on them.
- The Chanteuse: Frenchy is the singer in the saloon; Marlene Dietrich sings a couple of songs.
- Corrupt Hick: Kent, who is cheating people out of their land so he can buy up the whole valley and charge extortionate prices on anyone driving their cattle through it.
- Corrupt Politician: Slade, the slimy mayor who is in cahoots with Kent.
- Determined Homesteader's Wife: Mrs. Claggett, who reacts to Kent trying to take her farm by taking a shot at him with her rifle and yelling "Come and get it!"
- Famous Last Words: "That Gyp has kept me awake too much." - Wash
- Fixing the Game: Kent, with Frenchy's help, routinely cheats at poker in order to scam people out of their land.
- Funny Foreigner: Stavrogin, the eager Russian special deputy.
- High-HeelFace Turn: Frenchy falls in love with Destry, which leads to her calling him away from the attack on the jail, and eventually causes her to help the good guys at the end.
- Improbable Aiming Skills: Mild-mannered Destry proves a point about himself by borrowing a mook's gun and shooting all the knobs off a decorative wagon wheel. The intimidated mook then promises to stop making trouble.
- Let Me Tell You a Story: Destry is constantly telling "I had this friend" stories to defuse tension and make a point.
- Let's Get Dangerous!: Destry is mild-mannered and affable and initially appears to be a complete patsy, but as eventually becomes clear he is determined to take Kent and his gang down. Eventually he marshals a posse to go after Kent.
- Male Gaze: One card player can't help staring at Frenchy's bottom during the game.
- Match Cut: Between Frenchy doing her makeup in a mirror and Janice doing the same thing.
- Malicious Misnaming: Boris Stavrogin is called "Callahan" by everybody, because Lily Belle's first husband was named Callahan. Eventually, after trashing the portrait of Callahan that hangs in their room and putting up his own, he gets Lily Belle to admit that she is Mrs. Boris Stavrogin.
- Ms. Fanservice: Frenchy spends pretty much the whole movie in a succession of corsets.
- Redemption Equals Death: Frenchy.
- She's Got Legs: Dietrich was famous for her legs, and they are on display in hose throughout this film.
- Something Else Also Rises: As French is singing a number onstage, her admirer Boris is watching and holding a pack of cards, which eventually spurt out of his hand in a suggestive manner.
- Taking the Bullet: Accidentally, but Frenchy steps in to save Destry from Kent and gets fatally shot.
- Title Drop: "And when he gets here, Destry will ride again."—Wash
- Violence Really Is the Answer: Played with. Destry explains his more peaceful, non-confrontational style of law enforcement by stating that his father always wielded a gun and that didn't stop him from getting shot In the Back. However, later when Wash is murdered, Destry snaps and gathers a posse, intending to take Kent and the saloon by storm. Eventually, this trope is ultimately averted when the women of the town, determined to stop the impending bloodbath, invade Kent's saloon wielding two-by-fours. The only casualties are Frenchy and Kent.
- Victoria's Secret Compartment: When Kent snatches the deed away from her, Frenchy puts a chip from the game in her Compartment and says she'd rather have "money in the bank." When she goes home after a shift at the saloon she leans forward and spills an impressive amount of coins from the Compartment onto her dresser.