Sexual selection is an important force driving the evolution of morphological and genetic traits. To determine the importance of male–male, postcopulatory sexual selection in natural populations of house mice, we estimated the frequency of multiple paternity, defined as the frequency with which a pregnant female carried a litter fertilized by more than one male. By genotyping eight microsatellite markers from 1095 mice, we found evidence of multiple paternity from 33 of 143. Evidence for multiple paternity was especially strong for 29 of these litters. Multiple paternity was significantly more common in higher-density vs. lower-density populations. Any estimate of multiple paternity will be an underestimate of the frequency of multiple mating, defined as the frequency with which a female mates with more than a single male during a single oestrus cycle. We used computer simulations to estimate the frequency of multiple mating, incorporating observed reductions in heterozygosity and levels of allele sharing among mother and father. These simulations indicated that multiple mating is common, occurring in at least 20% of all oestrus cycles. The exact estimate depends on the competitive skew among males, a parameter for which we currently have no data from natural populations. This study suggests that sperm competition is an important aspect of postcopulatory sexual selection in house mice.