Here we use amplified fragment length polymorphism (AFLP) to assess genetic differentiation of Helicoverpa armigera and H. assulta. The results indicated that both species-specific fingerprints and cluster analysis showed the ability of AFLP technique to discriminate the two sibling species; among a total 1963 AFLP markers amplified from nine primer combinations: 777 (39.6%) were H. armigera-specific,602 (30.7%) were H. assultaspecific,and 584 (29.7%) were common bands. The mean number of H. armigera-specific bands was significantly more than that of H. assulta-specific bands for nine primer combinations (P＜0.05); the intraspecific distance of H. armigera and H. assulta was 0.123 0 and 0.110 7 respectively,and the interspecific distance was 0.178 3. In addition,the percentage of polymorphic loci and estimated average heterozygosity were used to estimate genetic diversity of the two species. This study therefore demonstrates that AFLP analysis is a sensitive and reliable technique to study genetic differentiation and genetic relationships between species and provides sufficient molecular markers for future linkage map construction,location and eventual cloning of genes involved in traits differentiation.