Entomopathogenic nematodes of the genus Heterorhabditis are morphologically conservative, consequently the majority of isolates remain unassigned at the species level. When a Heterorhabditis infective juvenile infects an insect host, it develops into a protandrous hermaphrodite female. These first-generation females give rise to a second generation which contains both males and females. Because of this complex life-cycle and also because of uncertainty as to whether second-generation females are amphimictic, cross-breeding studies to facilitate species determination have not been carried out previously. We demonstrate here that second-generation Heterorhabditis females are amphimictic. Because of this finding, we have been able to develop a successful cross-breeding technique for the purposes of species determination in Heterorhabditis. Interstrain crosses using second-generation males and females from the appropriate strains have been successfully set up in Xenorhabdus luminescens-treated G. mellonella cadavers and also on agar plates. Using the techniques described here we confirm that H . bacteriophora (Brecon strain), H . megidis and H . zealandica are distinct biological species, we note that the H. bacteriophora group contains at least 2 species and we provide evidence for the existence of a new Irish species of Heterorhabditis.