The bacterial symbiont of the entomopathogenic nematode Heterorhabditis bacteriophora strain GPS11 was characterized by 16S rRNA gene sequence and physiological traits. The phylogenetic tree built upon 16S rRNA gene sequences clustered the GPS11 bacterial isolate with Photorhabdus temperata strains which have been previously isolated from Heterorhabditis species. The phylogenetic tree further identified four subgroups in P. temperata, and the relationships among these subgroups were confirmed by gyrase subunit B (gyrB) gene sequence analysis. The subgroup containing the GPS11 bacterial isolate differs from other subgroups in sequences of 16S rRNA and gyrB gene, physiological traits, nematode host species, and geographic origin. Therefore, the subgroup comprising the GPS11 bacterial isolate is proposed here as a new subspecies: Photorhabdus temperata subsp. stackebrandtii subsp. nov. (type strain GPS11). The type strain has been deposited in ATCC and DSMZ collections.