Even though premating isolation is hypothesized to be a major driving force in speciation, its genetic basis is poorly known. In the noctuid moth Heliothis subflexa, one group of sex pheromone components, the acetates, emitted by the female, plays a crucial isolating role in preventing interspecific matings to males of the closely related Heliothis virescens, in which females do not produce acetates and males are repelled by them. We previously found intraspecific variation in acetates in H. subflexa: females in eastern North America contain significantly more acetates than females in Western Mexico. Here we describe the persistence of this intraspecific variation in laboratory-reared strains and the identification of one major quantitative trait locus (QTL), explaining 40% of the variance in acetate amounts. We homologized this intraspecific QTL to our previously identified interspecific QTL using restriction-associated DNA (RAD) tags. We found that a major intraspecific QTL overlaps with one of the two major interspecific QTL. To identify candidate genes underlying the acetate variation, we investigated a number of gene families with known or suspected acetyl- or acyltransferase activity. The most likely candidate genes did not map to our QTL, so that we currently hypothesize that a transcription factor underlies this QTL. Finding a single, large QTL that impacts variation in pheromone blends between and within species is, to our knowledge, the first such example for traits that have been demonstrated to affect premating isolation.