Variation among males and females in reproductive success is a major determinant of effective population size. Most studies of male mating success in Drosophila, however, have been done under conditions very different from those in typical cultures. We determined the distribution of male mating success in five laboratory populations of D. melanogaster maintained on a 14 d, discrete generation cycle fairly representative of standard Drosophila cultures. Mating success was measured as the number of matings a male could achieve under conditions closely approximating a regular culture vial of these populations. Preliminary studies determined that most mating in these populations occurred within 14 h of the flies attaining sexual maturity. Consequently, individual virgin males were marked with white paint on their thorax, put into vials with varying numbers of unmarked virgin flies of both sexes, and monitored continuously for matings over a period of up to 14 h. At various times during the assay, virgin males and females were added to these vials in proportions simulating the pattern of eclosion in culture vials. The observed variation in the number of matings per male in the five populations was, by and large, consistent with a Poisson distribution, suggesting that male mating success in short-generation-time, discrete-generation laboratory cultures of D. melanogaster may fulfil a fundamental assumption of the Wright–Fisher model of genetic drift in finite populations.