Male dimorphism of the subfamily Ostertagiinae (Nematoda: Trichostrongylidae) is a well-known phenomenon, and two or more morphotypes of a single species have previously been described as different species. Two Spiculopteragia spp., S. houdemeri (syn. S. yamashitai) and S. andreevae (syn. Rinadia andreevae) recorded in Asian cervids and wild bovids, are considered to represent major and minor morphs of S. houdemeri, respectively, based solely on their co-occurrence in the same host individual along with monomorphic females. In this study, males of morph houdemeri ( = S. houdemeri) and morph andreevae ( = S. andreevae) as well as females with three different vulval ornamentations were collected from sika deer (Cervus nippon) and Japanese serows (Capricornis crispus) distributed on the mainland of Japan. Morphologically characterized worms were subjected to molecular genetic analyses based on the internal transcribed spacer region of the ribosomal RNA gene and a partial region of the cytochrome c oxidase subunit I gene of mitochondrial DNA. Of 181 collected sika deer, 177 (97.8%) and 73 (40.3%) deer harboured males of morphs houdemeri and andreevae, respectively. Worm numbers of the former morph were found to range between 1 and 444 per individual, whereas only 1–25 worms per individual were detected for the latter morph. Five out of six serows harboured 47–71 or 2–9 males of morphs houdemeri and andreevae per individual, respectively. Females with one or two vulval flaps were predominant, but there was a substantial presence of flapless females in both host species. All the morphs of male and female adults had an identical genetic background, thus directly confirming the morphological polymorphism of S. houdemeri.