Behavioral genetic mapping studies in model organisms predominantly use crosses originating from a single pair of inbred lines to determine the location of alleles that confer genetic variation in the trait of interest, and they often make sweeping generalizations about the genetic architecture of the trait based on these results. A previous study fine mapped mate preference variation between one pair of Drosophila pseudoobscura lines and identified 2 strong-effect behavioral quantitative trait loci (QTLs). Here, we replicated the previous study's mapping design to examine the extent of variation at these behavioral QTLs across 6 pairs of lines, but we were unable to detect effects of either QTL region in the pairs of lines studied. We suggest that the low-discrimination alleles at these 2 QTLs may occur at low frequency within D. pseudoobscura, although other explanations for the inconsistency are possible. These results underscore the need to examine multiple strains across a species when describing the genetic variation underlying behavioral traits.