Recent experiments with Drosophila have demonstrated that the success of sperm in multiply mated females depends on the genotype of both the male and the female. To further characterize the distinction between male and female roles in sperm success, we scored variation in both sexes in sperm competitive ability among a set of chromosome replacement lines that allow identification of effects to each chromosome. We detected significant male and female effects on sperm precedence, defined as the ability of a male's ejaculate to displace resident sperm (P2) or avoid being displaced by subsequent matings (P1). Tests of effects of first, second and third chromosome substitutions revealed significant differences among third chromosomes in male sperm precedence (both P1 and P2) and a first × second chromosome interaction in female's effect on sperm precedence (only P1). We found no significant correlation between male and female effects on sperm precedence, suggesting that the variation found in both P1 and P2 has a different genetic cause in the two sexes.