Abstract We applied association analysis to elucidate the genetic basis for variation in phenotypes affecting postcopulatory sexual selection in a natural population of Drosophila melanogaster. We scored 96 third chromosome substitution lines for nine phenotypes affecting sperm competitive ability and genotyped them at 72 polymorphisms in 13 male reproductive genes. Significant heterogeneity among lines (P < 0.01) was detected for all phenotypes except male-induced refractoriness (P = 0.053). We identified 24 associations (8 single-marker associations, 12 three-marker haplotype associations, and 4 cases of epistasis revealed by single-marker interactions). Fewer than 9 of these associations are likely to be false positives. Several associations were consistent with previous findings [Acp70A with the male's influence on the female's refractoriness to remating (refractory), Esterase-6 with a male's remating probability (remating) and a measure of female offspring production (fecundity)], but many are novel associations with uncharacterized seminal fluid proteins. Four genes showed evidence for pleiotropic effects [CG6168 with a measure of sperm competition (P2′) and refractory, CG14560 with a defensive measure of sperm competition (P1′) and a measure of female fecundity, Acp62F with P2′ and a measure of female fecundity, and Esterase-6 with remating and a measure of female fecundity]. Our findings provide evidence that pleiotropy and epistasis are important factors in the genetic architecture of male reproductive success and show that haplotype analyses can identify associations missed in the single-marker approach.