A new species of entomopathogenic nematode, Steinernema khoisanae n. sp. is described from South Africa. The new species is characterised by morphometrics of the infective juvenile with body length 1076 mu m, narrow body diameter of 33 mu m, excretory pore 94 mu m from anterior end, tail 85 mu m long, a = 33, D% = 68, H% = 57, and E% = 111. The lateral field pattern of the new species is 2, 7, 8, 6, 4 and 2. The male of the first generation can be recognised by the spicule and the gubernaculum shape, excretory pore located posteriorly near the end of the pharynx, D% = 88, and SW% = 199. The first generation female can be recognised by the non-protruding vulva and tail bearing a prominent mucron. Steinernema khoisanae n. sp. is characterised genetically by sequences of the internal transcribed spacers and D2/D3 regions of 28S ribosomal DNA, by composition of their sequences and by numerous unique, derived, nucleotide character states. Phylogenetic trees show that S. khoisanae n. sp. and other members of the S. glaseri-group form a monophyletic assemblage.