SUMMARY Strongyloides venezuelensis is a parasitic nematode that infects rodents. Although Strongyloides species described to date are known to exhibit parthenogenetic reproduction in the parasitic stage of their life cycle and sexual reproduction in the free-living stage, we did not observe any free-living males in S. venezuelensis in our strain, suggesting that the nematode is likely to depend on parthenogenetic reproduction. We confirmed by cytological analysis that S. venezuelensis produces eggs by parthenogenesis during the parasitic stage of its life cycle. Phylogenetic analysis using nearly the full length of 18S and D3 region of 28S ribosomal RNA gene suggested that S. venezuelensis is distantly related to another rodent parasite, namely Strongyloides ratti, but more closely related to a ruminant parasite, Strongyloides papillosus. Karyotype analysis revealed S. venezuelensis reproduces with mitotic parthenogenesis, and has the same number of chromosomes as S. papillosus (2n = 4), but differs from S. ratti (2n = 6) in this regard. These results, taken together, suggest that S. venezuelensis evolved its parasitism for rodents independently from S. ratti and, therefore, is likely to have a different reproductive strategy.