Female Drosophila melanogaster frequently mate with multiple males in nature as shown through parentage analysis. Although polyandry is well documented, we know little about the timing between mating events in wild Drosophila populations due to the challenge of following behaviours of individual females. In this study, we used the presence of a male reproductive protein that is transferred to the female during mating (Sex Peptide, SP) to determine whether she had recently mated. We sampled females throughout the day, conducted control matings to determine the decay rate of SP within the female reproductive tract and performed computer simulations to fit the observed proportion of mated females to a nonhomogenous Poisson process that defined the expected time between successive matings for a given female. In our control matings, 100% of mated females tested positive for SP 0.5 h after the start of mating (ASM), but only 24% tested positive 24 h ASM. Overall, 35% of wild‐caught females tested positive for the presence of SP. Fitting our observed data to our simple nonhomogenous Poisson model provided the inference that females are mating, on average, approximately every 27 h (with 95% credibility interval 23–31 h). Thus, it appears that females are mating a bit less frequently that once per day in this natural population and that mating events tend to occur either early in the morning or late in the afternoon.