The cry1-type genes of Bacillus thuringiensis represent the largest cry gene family, which contains 50 distinct holotypes. It is becoming more and more difficult to identify cry1-type genes using current methods because of the increasing number of cry1-type genes. In the present study, an improved PCR-restriction fragment length polymorphism (PCR-RFLP) method which can distinguish 41 holotypes of cry1-type genes was developed. This improved method was used to identify cry1-type genes in 20 B. thuringiensis strains that are toxic to lepidoptera. The results showed that the improved method can efficiently identify single and clustered cry1-type genes and can be used to evaluate cry1-type genes in novel strain collections of B. thuringiensis. Among the detected cry1-type genes, we identified four novel genes, cry1Ai, cry1Bb, cry1Ja, and cry1La. The bioassay results from the expressed products of the four novel cry genes showed that Cry1Ai2, Cry1Bb2, and Cry1Ja2 were highly toxic against Plutella xylostella, whereas Cry1La2 exhibited no activity. Moreover, Cry1Ai2 had good lethal activity against Ostrinia furnacalis, Hyphantria cunea, Chilo suppressalis, and Bombyx mori larvae and considerable weight loss activity against Helicoverpa armigera.