The current study evaluates the potential of using high resolution DNA melting assays to discriminate species in the genus Isaria. The study utilizes a previously identified 103 base pair PCR amplicon, which was reported to be selective for Isaria fumosorosea. Our study finds the amplicon selective for Isaria javanica and Isaria poprawskii when assayed against all members of the genus. In addition, the high resolution melting profile of this amplicon can be used to discriminate between I. javanica, I. poprawskii and a 1:1 mixture of the two species. The practical application of this technique was confirmed using a bioassay on whitefly nymphs (Bemisia tabaci biotype B) inoculated with I. javanica, I. poprawskii or a 1:1 mixture of the two species. This assay provides a simple assay to identify these two species of entomopathogenic fungi.