Although male courtship songs have been repeatedly implicated in sexual isolation between numerous Drosophila species, no genetic studies have evaluated the genetic basis of differences between species beyond using quantitative genetic analyses of hybrids or surveying associations of song characters to five or fewer genetic markers. Here, we dissect the genetic basis of the difference between D. pseudoobscura and D. persimilis in two courtship song elements (interpulse interval and intrapulse frequency) using 15 molecular markers. We also evaluate the association between song elements and sexual isolation in these backcross hybrid males of these species. We find that song differences between these species are associated with at least two or three genomic regions, and the species difference in interpulse interval may be oligogenic. Courtship song differences are especially strongly associated with two inversions that differentiate these species. Further, we found that interpulse interval is strongly associated with mating success to D. pseudoobscura females, while intrapulse frequency is associated with mating success to D. persimilis females. Implications of these findings are discussed.