Restriction digests of amplified DNA from the mitochondria1 genome and the nuclear ribosomal internally transcribed spacer region have been evaluated as genetic markers for species groups in Heterorhabditis. Six RFLP profiles have been identified. These profiles supported groupings determined by cross-breeding studies and were in agreement with less definitive groupings based on other biochemical and molecular methods. Digestion patterns of both amplification products provided strong evidence for the recognition of species groups, which include Irish, NW European, tropical, and a H . bacteriophora complex. The H . bacteriophora complex could be further resolved into three genotypes represented by H . zealandica, the H . bacteriophora, Brecon (Australian) type isolate for H . bacteriophora, and a grouping composed of isolates NCl, V16, HI82, and HP88. All cultures obtained of the H . megtdis isolate were identical to the NW European group. These results could be used to aid monitoring of field release of Heterorhubditk as well as allowing a rapid initial assessment of taxonomic grouping.