All known species of Fergusonina flies (Fergusoninidae) participate in an obligate mutualism with Fergusobia nematodes (Neotylenchidae). From dissections, it is believed that all female third-instar larvae, pupae and adult female flies carry nematodes internally, while male adults and larvae do not. This observation of sex-limited association was tested using a molecular diagnostic approach. Nematode-specific primers were used for polymerase chain reaction amplification of a portion of the mitochondrial cytochrome oxidase I gene in order to screen DNA extractions from flies for the presence of nematode DNA. Consistent with evidence from dissections, nearly all extractions from adult female flies were positive for nematode DNA, while all DNA extractions from adult male flies were negative. Nearly half of late-instar larvae screened showed evidence of nematode DNA, consistent with the hypothesis that nematodes are only present within females. This paper is the first to use a molecular method to confirm this phenomenon within the Fergusonina/Fergusobia system.