An open question in evolutionary biology is the relationship between standing variation for a trait and the variation that leads to interspecific divergence. By identifying loci underlying phenotypic variation in intra- and interspecific crosses we can determine the extent to which polymorphism and divergence are controlled by the same genomic regions. Sexual traits provide abundant examples of morphological and behavioral diversity within and among species, and here we leverage variation in the Drosophila sex comb to address this question. The sex comb is an array of modified bristles or 'teeth' present on the male forelegs of several Drosophilid species. Males use the comb to grasp females during copulation, and ablation experiments have shown that males lacking comb teeth typically fail to mate. We measured tooth number in >700 genotypes derived from a multiparental advanced-intercross population, mapping three moderate-effect loci contributing to trait heritability. Two quantitative trait loci (QTLs) coincide with previously identified intra- and interspecific sex comb QTL, but such overlap can be explained by chance alone, in part because of the broad swathes of the genome implicated by earlier, low-resolution QTL scans. Our mapped QTL regions encompass 70-124 genes, but do not include those genes known to be involved in developmental specification of the comb. Nonetheless, we identified plausible candidates within all QTL intervals, and used RNA interference to validate effects at four loci. Notably, TweedleS expression knockdown substantially reduces tooth number. The genes we highlight are strong candidates to harbor segregating, functional variants contributing to sex comb tooth number.