Poison Ivy gets the cover for her big debut, in Batman 181 (June 1966), in a story by Kanigher, Moldoff and Giella.
Three other female villains get introduced in this story: Dragon Fly, Silken Spider and Tiger Moth. Each claims to be the “Queen of Crime” in Gotham, but Poison Ivy shows up to insist that only she deserves that title.
Ivy lures the three woman into a trap, using an electrified crown to hold them. Batman and Robin show up, and Ivy makes a play for Batman. She kisses him, but he rebuffs her. This story only really endows her with one power, that of being able to climb a building the way ivy does. Curiously, this is not a power that remains in her repertoire.
Batman captures her, but she vows to return, which she does in the next new issue (182 is an all-reprint issue). As for the other three women, they have to wait until the 90s to return.
This issue also contains the best Mystery Analysts story, possibly because it’s really more of a Batman story than anything else, by Fox, Moldoff and Greene..
Kaye Daye shows up for a meeting of the Analysts, announcing that her latest book was not written by her. Just after she explains this, her brooch relays a death threat.
Batman is very suspicious about all of this. He figures out that the Kaye Daye who came to the meeting could not have been the real one, and must have been in on the scam, in order to be wearing the correct brooch.
He pieces together some subtle but clear clues, and finds the real Kaye Daye, as well as her impersonator. As I said, the best of the Mystery Analysts series.