Steinernema feltiae and S. kraussei were isolated from Hokkaido, Japan. This is the first record of S. kraussei and the first definitive record of S. feltiae from Japan. The morphological variation of the infective juveniles and the first generation males of Japanese isolates of both species are reported. Intraspecific variation in the PCR-RFLP analysis of the ITS region of ribosomal DNA was observed in both species. The Japanese isolates of S. feltiae showed different RFLP patterns from European isolates with Dde I and Hinf I restriction digests. The Japanese isolate of S. kraussei also showed an RFLP variation from the UK and Russian isolates upon Dde I restriction digestion. Moreover, in each isolate of S. kraussei, some intra-population variations were observed with some restriction digestions. The intraspecific variation in the ITS region of the rDNA could be used as a molecular marker to distinguish the Japanese isolates from European isolates of S. feltiae if the latter was introduced as a biological insecticide.