Polymorphic genetic markers were identified and characterized using a partial genomic library of the tobacco budworm, Heliothis virescens (Fabricius), enriched for simple sequence repeats (SSR) and nucleotide sequences of expressed sequence tags (EST). Nucleotide sequences of 192 clones from the partial genomic library yielded 147 unique SSRs while EST evaluations identified 280 SSR-containing sequences. All anonymous SSRs and 192 EST-SSRs were evaluated to select loci that produced the best quality peaks without stutter peaks. Six anonymous markers and nine EST-derived markers were selected to evaluate a sample of 96 insects collected from Stoneville, MS. The observed number of alleles ranged from two to eight, with an average of 5.87 (SE ± 0.53). The polymorphic information content (PIC) value averaged 0.446 (SE ± 0.052). Significant deviations from Hardy-Weinberg expectations were detected at eight loci. We speculate that these loci are under selection in the population of tobacco budworm used in the study. No significant linkage disequilibrium was detected at any of the loci. Proportions of anonymous and EST-SSR markers validated after extensive evaluation were 4.08 and 4.68%, respectively. One anonymous SSR marker (HvMS117) and all nine EST markers were transferable to the bollworm, Helicoverpa zea (Boddie).