Evidence for multiple insemination in the mosquitofish, Gambusia affinis, was detected by statistical analysis of the enzyme phenotypes in mother-offspring combinations for three loci. The probability of nondetection of multiple insemination events for diallelic loci with two or more males mating with a female was presented. The frequency of multiple insemination for G. affinis is at least 56% but is probably nearer to 100%. The high incidence of multiple insemination together with sperm storage and delayed fertilization could have important effects on allele frequencies through time and space. These effects are due to the increase in effective population size and overlap among generations.