The Oscheius subgenus (Nematoda: Rhabditidae) comprises several common free-living hermaphroditic species. Morphological identification is difficult due to a lack of reliable characters to discriminate species. We studied 32 strains that are closely related to Rhabditis (Oscheius) pseudodolichura and R. (O.) tipulae. We present results from mating experiments between the strains and sequence data from the internal transcribed spacer region of ribosomal RNA, allowing discrimination of three closely related biological species.