[Objectives]  To establish a molecular technique that can reliably distinguish the cotton bollworm, the tobacco budworm, and hybrids between these species, to determine the effect of different kinds of light on hybridization, and the extent of hybridization in a mixed population under greenhouse conditions. [Methods]  Hybridization rates under all potential kinds of pairings between 3 day-old virgin cotton bollworm and tobacco budworm adults were recorded under a 0.5 lx black lamp, an incandescent lamp, and complete darkness (control). Microsatellite loci that could reliably distinguish cotton bollworms, tobacco budworms, and their hybrids, were identified and used to identify the resultant progeny. A mixed population of cotton bollworm and tobacco budworm adults were released in a greenhouse and their offspring screened using the previously identified microsatellite markers. [Results]  Hybridization could result from all kinds of forced pairings. However, in mixed populations, the proportion of hybrids was just 2.92%, and only one kind of pairing (male cotton bollworm × female tobacco budworm) was observed. There was no significant difference in hybridization rates between the black lamp, incandescent lamp, and dark, treatments. The sizes of the microsatellite loci HarSSR1、HarSSR9, and HarSSR10, differ between the two species. No hybrids were identified among 360 offspring screened during two years research on a mixed, greenhouse population. [Conclusion]  Hybridization between the cotton bollworm and tobacco budworm can occur when individuals are not free to choose mates but low intensity light does not promote hybridization. Only one kind of pairing was observed in mixed populations, suggesting that male cotton bollworms are competitively superior to male tobacco budworms in competition for mates. The cotton bollworm, tobacco budworm, and hybrids between these species, could be reliably distinguished with microsatellite markers, which provide a simple and accurate molecular identification method. Hybrids were not identified during two years study of a mixed, greenhouse population, indicating that there is premating reproductive isolation between the two species.