Spatial and temporal variation in the genetic structure of local populations of tobacco budworm Heliothis virescens (F.) were surveyed using 11 polymorphic allozyme and 36 random amplified polymorphic DNA (RAPD)-polymerase chain reaction markers. Allelic and genotypic frequencies were calculated from male moths caught in pheromone traps from 5 to 6 local populations from 1995 to 1997. Overall, populations showed little spatial differentiation that indicates a high effective gene flow rate over the spatial scale under investigation. The data show that allelic differentiation arose more frequently in the second generation than in any other generation, suggesting that dispersal was more limited during this period of the summer. Based on allozyme data, estimates of Wright’s standardized genetic variance among populations, F_ST, ranged from 0.001 to 0.018, with the highest values found from the second generation. Estimates based on bootstrapping over loci suggested that genetic variance among populations was significantly higher in the second generation than other generations. Genetic variance tended to increase progressively through the season, peaking in the second generation and decreasing in the latter part of the summer. This temporal pattern of genetic structure is concomitant with changes in gene flow rates and may be influenced by phenological changes in the primary host plants. The F_ST estimates and average heterozygosity from RAPD markers were consistently higher than those estimated from allozyme markers. However, patterns of change over the course of the summer were similar, and the two types of genetic markers provide similar insights into the dispersal biology of H. virescens.