“Oh, I love it when I’m nasty!”

@Mugen Kagemaru  
You get my point. It still exists and it reminds me enough of the One Ring that I don’t think it’s gonna be gone for good until it no longer exists at all.

@Scrounge
 
Actually, Zecora buried it somewhere.

@PonyPon  
I forgot about that, but still, Zecora’s hut isn’t exactly Fort Knox. If one of the writers decided to pull it back out, it would be simple enough to get it back in play.

@Scrounge  
It’s being held under lock and key by Zecora.

@VoltraTheLively  
So we’ve had one villain with no gender and one villain that could be considered both.
 
Speaking of the Alicorn Amulet, is anyone else kind of surprised that the ending of “Magic Duel” didn’t even bother to mention what they did with it after they got it off Trixie? You’d think they’d have the Mane Six go throw it into the fires of Mount Doom or something. At least the Inspiration Manifestation book was disposed of, assuming Spike’s digestive system works properly.

@Scrounge
 
Plants are more hermaphrodites than genderless, having male and female planty organs.

@Mugen Kagemaru  
Really? I thought that sort of thing would be on Wikipedia and the problem is me being too lazy to look it up.

@Scrounge
 
I really wish I could confirm that for you, but it’s kinda classified. Sorry.

OP has “asexual” and “genderless” confused.
 
And technically, we’ve already had a genderless antagonist: the Alicorn Amulet. Two if you count the black vines, but I’m only like 80% sure about plants not having genders.

Please be a dragon

@Background Pony #59D5  
That sounds really dirty out of context.

@Background Pony #A7A3  
The mistress?

@the metal bronyhead
 
No, no, no, and no!
 
Repeat after me!
 
“I, Garland, will KNOCK YOU ALL DOWN!”
 
Get it fucking right, you shit-spewing n00b.
 
[/sarcasm]
 
I’m genuinely surprised someone used the literal translation and not the popular one.

@heem an  
Ahuizotl is, actually.

ahuizotl and maniac are not real villains

@Anonymous444444444  
Chimera and Diamond Dogs also missing. And Trixie (or just the Alicorn Amulet itself). There’s probably more, too.

@621Chopsuey  
Say whatever about his plan, this guy’s crazy prepared.

@Background Pony #A7A3
 
Like Master Xehanort.
 
Find some poor saps to become his vessels of darkness, plunge a few worlds (a la Equestria) into darkness in his pursuit of the X-blade, and also fits the remaining four criteria.
 

 
Big Kingdom Hearts fan, regret nothing.

@the metal bronyhead  
Oh, dreidel, dreidel, dreidel…I made you out of (polished steel)…Dreidel, dreidel, dreidel…with dreidel I shall slay.

Well let’s see, its been female to male trade off so far, so either our next three villains are male or we get three males at once. That is assuming it follows this trend.

Asexual of course…
 
Who else but THE SMOOZE

@Anonymous444444444  
That’s his weapon

@the metal bronyhead  
Why is he carrying a giant metal dreidel?

@the metal bronyhead  
Reply with Kefka Palazzo, and the show already has both Discord and Mane-iac.  
Rugal Bernstein = Tirek  
The Borg Queen = Queen Chrysalis  
NO Shounen Jump examples, please.
 
Since Lauren Faust-McCracken is a Whovian, why not a Time Lord/Lady, only antagonistic?