Acoustic signals often play a significant role in pair formation and in species recognition. Determining the genetic basis of signal divergence will help to understand signal evolution by sexual selection and its role in the speciation process. An earlier study investigated QTL for male courtship song carrier frequency in Drosophila montana using microsatellite markers. We refined this study by adding to the linkage map markers for ten candidate genes known to affect song production in D. melanogaster. We also extended the analyses to additional song characters (pulse train length, pulse number, interpulse interval, pulse length and cycle number). Our results indicate that loci in two different regions of the genome control distinct features of the courtship song. Pulse train traits (pulse train length and pulse number) mapped to the X chromosome, showing significant linkage with the period gene. In contrast, characters related to song pulse properties (pulse length, cycle number and carrier frequency) mapped to the region of chromosome 2 near the candidate gene fruitless, identifying these genes as suitable loci for further investigations. In previous studies, the pulse train traits have been found to vary substantially between Drosophila species, and so are potential species recognition signals, while the pulse traits may be more important in intra-specific mate choice.