The bacterial symbionts SF41(T) and SF783 were isolated from populations of the insect pathogenic nematode Heterorhabditis zealandica collected in South Africa. Both strains were closely related to strain Q614 isolated from a population of Heterorhabditis sp. collected from soil in Australia in the 1980s. Sequence analysis based on a multigene approach, DNA-DNA hybridization data and phenotypic traits showed that strains SF41(T), SF783 and 0614 belong to the same species of the genus Photorhabdus with Photorhabdus temperata subsp. cinerea as the most closely related taxon (DNA DNA hybridization value of 68%). Moreover, the phylogenetic position of Photorhabdus temperata subsp. cinerea DSM 19724(T) initially determined using the gyrB sequences, was reconsidered in the light of the data obtained by our multigene approach and DNA-DNA hybridization experiments. Strains SF41(T)., SF783 and 0614 represent a novel species of the genus Photorhabdus, for which the name Photorhabdus heterorhabditis sp. nov. is proposed (type strain SF41(T)=ATCC BAA-2479(T)=DSM 25263(T)).