At the conclusion of the 'Princess ponies' episode, all of the Princess ponies (after fighting over who should be queen) finally compromise and decide to 'takes turns' being Queen (haha) So I guess they are all sometimes-queens? (Tiffany, Sparkle, Serena, Royal Blue, Primrose, whoever I've forgotten)
As others have said, Rosedust introduces herself as Queen of the Flutter ponies and Majesty is the queen of Pony land:) Majesty came with the castle (it's hers!) and there are G1 annuals and tape-recordings refering to her as the Queen.
Can't think of G1 Queen villains right now, then again I haven't seen every episode!
In that episode about the newborn twins, the ponies are invited to a party by an evil 'Duchess'. Turns out she is an evil servant that stole the throne, and the rightful Duchess is hiding or imprisoned in the palace or something.
Um, there is the King of the Grundles and Lavan the king of demonic-looking-lava-people (but ok they are not queens).