We only saw Lady Tremaine and two step-sisters in the episode The Other Show, neither one was named Anastasia. Plus Lady Tremaine took one of them with her to The Land of Untold Stories but I'm guessing Ana going to Wonderland happened before the ball. Her mother likely favored Clorinda over the Tisbe and Ana. Anastsia was never able to gain her mother's approval and simply wanted to be happy. Clorinda even said that she had to be cruel to Cinderella to keep her mother's approval, so Ana likely wasn't cruel to her and her mother was disappointed in her, so that gave Clorinda motivation to treat Cinderella badly (even though she didn't want to). Ana knew she never would get the Prince with her mother favoring her sister over her, so she gave up and found Will. Her mother finally caught up with her when she was about to leave for Wonderland, which I believe happened before the events of the Cinderella story. Ana's mother says herself that at least she still has Ana's sisters. I doubt she'd be referring to Cinderella in that plural. If she is Cinderella's step-sister, it has been shown that Cinderella has more than two step-sisters. Ana likely became jealous of Cinderella from going to a servant that her mother looked down upon to becoming a princess. True, Ana was a queen at that point, but maybe if she had stayed she could've gotten the prince and her mother's love. The Jabberwocky did say that Ana had a step-sister that married a prince, which seems like MUCH more than just a coincidence. She has to be Anastasia Tremaine, Cinderella's step-sister.

1.Both are named Anastasia
2.They both have overly-controlling mothers that prefer moving up in life over love and are disappointed in their daughter named Anastasia
3.They both have a step-sister that married a prince

WHO THE HELL ELSE COULD SHE BE! I find it impossible to believe that Once Upon A Time, which is owned by Disney, and includes Disney characters in its show has a character named Anastasia with that many similarities to Anastasia Tremaine isn't Cinderella's step-sister. The writers may have said on Twitter that she's not Cinderella's step-sister, but that doesn't mean much. I remember back when season 2 was getting close to being finished and they said that Peter Pan would NOT be appearing in the show AT ALL. They didn't said he wouldn't appear in season 2, they said that he wouldn't appear in the show. I think that shows that they're not always telling the truth because they don't wanna give away any surprises. They said they hope to tell her story in season 6 and this may be their way of keeping it a secret. But until they have Ana and Cinderella together face-to-face and say that they're not related, I refuse to believe that they're not step-sisters. I mean, it's pretty obvious!

Personally I've opted to just ignore 6x03 entirely, but this is a good theory. I got the feeling from watching the episode that the character of Clorinda was originally written to be Anastasia, but they couldn't get Emma Rigby and/or Michael Socha back, and they didn't want to recast them for the same reason they didn't want to recast Jefferson, so they altered the story a tiny bit and pretended they hadn't made Ana out to be Cinderella's stepsister in OUATIW to begin with.

These writers don't know their own goddamn continuity. The evidence is ALL THERE.

1) Anastasia's last name is Tremaine
2) The stepfamily's last name is Tremaine
3) It's mentioned in OUaTiW that Anastasia's stepsister married the prince.

This will probably be one of those things that will never be addressed or mentioned ever again (like the fact that Aurora and Philip still don't know that Maleficent is in Storybrooke, or Regina's treatment of Graham). To make it worse, I think Adam and Eddy have confirmed that Anastasia has nothing to do with Cinderella or the stepfamily.
About the Peter Pan thing, this means that they're lying or trolling the fans. Which makes them no better than awful writers and producers like I. Marlene King (of Pretty Little Liars infamy). That, or Anastasia truly has no connection to Cinderella whatsoever. Either way, it's not cool at all.