The predatory mites, Neoseiulus longispinosus (Evans) and N. womersleyi (Schicha) (Phytoseiidae) are two morphologically close species. Although they can be mainly separated by the length of the dorsal seta S5, misidentifications may occur. The present study investigated various strains of the two closely related species collected from Japan, Taiwan and/or Thailand using a combination of morphological and molecular data. We described and illustrated the adults and immature stages. We also sequenced the 28S region of nuclear ribosomal DNA to determine whether these strains represent different species. The two species could be clearly separated based on the length of seta S5 and the ratio of setae Z5/S5. In a 28S tree, the examined strains also formed separate clades with 100% bootstrap values. We conclude that a combination of different methods is often necessary to precisely distinguish these closely related taxa.