Queen Penelobee (Catherine Disher) is a clone of Penelope, created from a mix of alien bee and human DNA. After the death of their previous queen, the alien bee people of Droneopolis decided to choose a new queen from the "most evil female" in the cosmos; they had concluded that this person was Penelope. Penelope was abducted, but was later rescued by Betty and her crew; all the while, Penelope had thought the incident was only a dream. However, the bees of Droneoplis had managed to retain a strand of Penelope's hair. DNA was used from the hair, along with some of their own DNA to create a hybrid clone of Penelope as their new queen.
Queen Penelobee is similar to Penelope in most aspects, but is unfamiliar with Betty (ironic as Betty and Penelope interact regularly) and has romantic feelings for Sparky, after he had posed as a "king bee" in "Bee Movie". It is only in "Queen For a Day" that Queen Penelobee and Penelope meet in person (though Penelope believes the bees to be part of a virtual reality game); the bees of Droneopolis were willing to obey both due to their common hatred of Betty.