Females of the tobacco budworm, Heliothis virescens (F.), exhibit distinct geographical and temporal variation in sex pheromone composition, but the causes and significance of this variation are largely unexplored. Here we assessed whether 1) female pheromone variation was related to the host plants of origin, and 2) pheromone lures with varying amounts of Z9–14:Ald or 16:Ald were differentially attractive to males. Variation in female pheromone did not seem to be related to the host plants from which the eggs or larvae were collected, which may be because field-collected larvae were reared for three to five larval stages on artificial diet. By varying the concentration of Z9–14:Ald within the range in the female pheromone gland, we found males were more attracted as the amount increased from 1 to 10% relative to Z11–16:Ald, but significantly less with the highest concentration of 25%. In contrast, with 16:Ald, similar numbers of tobacco budworm males were caught in all traps where 16:Ald ranged from 0 to 200%. These results show that variation in Z9–14:Ald but not 16:Ald is evolutionary significant and likely subject to stabilizing selection in the field.