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Miss Yvonne (real first name: Yvonne) was a frequent visitor to the playhouse; obsessed with fashion and beauty; commonly proclaimed as "The Most Beautiful Woman in Puppetland" despite some casual viewers thinking the opposite of her beliefs, although Pee-Wee and his Playhouse characters such as Chairry often agree with her views of being the most beautiful (probably to be polite). She is usually dressed in gaudy prom/bridesmaid-type gowns with many petticoats and crinolines, has her hair in a bouffant, and wears white high heels. She is often paired with Cowboy Curtis, who does things together with Pee-Wee, and in one episode asked out Cowboy Curtis on a date, where Pee-Wee helped Cowboy Curtis prepare for it by first going on a practice run with Cowntess. Despite this, she had crushes on a lot of men during the show, including Conky Repairman Johnny Wilson, Tito, Ricardo and Fireman Frank, though she never pursued a committed romantic relationship with any of these men.

In Ants in Your Pants, she calls Conky her favorite robot.

In the episode Miss Yvonne's Visit, it is revealed to us that while she has great beauty and a warmhearted image, Yvonne actually is a terrible, slovenly houseguest. She also has several moving men/bodyguards employed in her service (including a man resembling Ricardo who is played by actor Vic Trevino), and whenever she travels anywhere she literally takes her makeup and beauty supplies with her to maintain her famous beauty.

The character was played by Lynne Marie Stewart, who had also worked with Paul Reubens in the film Pee-Wee's Big Adventure, although not as Miss Yvonne. In the film, she played an actress who was frustrated at trouble on the set over a pretentious child actor who has Pee-Wee's bike. Another film she did was the 1987 film The Running Man, starring Arnold Schwarzengger. Somewhat akin to Miss Yvonne, Stewart played an audience member who gushed over the host and selected a hockey-themed "Stalker" named "Subzero" to try and kill Arnold. Subzero was played by Professor Toru Tanaka, who also played Francis' butler in Pee-Wee's Big Adventure.

The character first appeared in The Pee-wee Herman Show, also played by Stewart.

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