Mitochondrial DNA (mtDNA) restriction-site variability was surveyed in geographically divergent collections of tobacco budworm, Heliothis virescens (F.). Two types of mtDNA analysis were employed, total DNA was digested with restriction enzymes and Southern blots probed with labeled purified mtDNA, and polymerase chain reaction amplified portions of the mtDNA (16S ribosomal DNA and the COI-COII regions) were subjected to digestion with restriction enzymes and the fragments visualized directly. Individuals from four locations in the United States and Mexico were used (Georgia, California, and two in Sonora). Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was also used on a smaller number of individuals from Tennessee, Mississippi, Oklahoma, and Texas. For the total mtDNA, 11 restriction enzymes cleaved ≈65 sites. Seven restriction enzymes cleaved ≈30 restriction sites using PCRRFLP. Seven haplotypes were identified with each technique. Haplotype 1 (total mtDNA) comprised 85% of individuals. The other six haplotypes were found only once or twice. Haplotype 1 (PCR) was found in 91% of the individuals. The other five haplotypes were rare. Population distributions show that the rare haplotypes are scattered among the geographical locations. No major geographically distinct populations were discovered. This suggests that the overall population passed through a bottleneck and expanded from a small population in recent evolutionary history, or that it annually expands from a common reservoir population, or that incoming migrants breed freely with those that may have overwintered locally, or a combination of the three. The findings are consistent with the reported migratory nature of the species, distribution of morphometric types in the western hemisphere, and a survey of enzymatic polymorphisms in the southern United States.