During a reproductive cycle, not all daughter mites of Varroa destructor mate and thus leave the brood cells as virgins. Here, we show that virgin mites are present within both the phoretic (10%) and reproductive (8%) mite population. Most (n = 29 of n = 33) of these encountered virgins laid unfertilized (= male) eggs, and some (n = 10) mated later on with their own son. These findings were verified by tests with artificially reared virgin mites. Obviously, mating is not a prerequisite for Varroa reproduction. However, due to the small number of reproductive cycles, the contribution of virgins to the Varroa population is regarded as low. This study also confirms conclusively that sex of V. destructor is determined via arrhenotokous parthenogenesis and not-as previously assumed-via pseudo-arrhenotoky. Furthermore, reproductive parameters of naturally invaded and artificially introduced Varroa females were compared, and artificial infestation was reconfirmed as a suitable method.