The tobacco Nicotiana rustica is widely used as a trap crop in the fields of Nicotiana tabacum in China, by attracting oviposition of Helicoverpa assulta females, thus preventing damage to N. tabacum. The mechanism underlying the differential oviposition rates of H. assulta across these two tobacco species, however, is largely unknown. We investigated the mechanism of host plant acceptance of H. assulta with respect to these two tobaccos by using a two-choice behavioral bioassay and GC–MS. Our results indicate that both the leaves and inflorescences of N. rustica attracted significantly more eggs than the corresponding parts of N. tabacum. Extracts of leaves and inflorescences of N. rustica with two different solvents elicited similar oviposition patterns to the corresponding parts of the plants. Chemical analysis by GC–MS revealed that the volatiles of N. rustica contain larger amounts of nicotine than those of N. tabacum at the flowering stage. In addition, γ-terpinolene and β-elemene are found only in extracts of N. rustica. A two-choice bioassay on the individual compounds showed that γ-terpinolene, which is specific to the vegetative stage of N. rustica, and nicotine attracted oviposition by H. assulta. The volatile β-elemene, which is present only in N. rustica, was also attractive. We conclude that the larger amount of nicotine, and the species-specific γ-terpinolene and β-elemene may mediate the different oviposition rates of H. assulta females across N. rustica and N. tabacum.