Nematodes of the family Heligmonellidae (Heligmosomoidea; Trichostrongylina) reside in the digestive tracts of rodents and lagomorphs. Although this family contains large numbers of genera and species, genetic information on the Heligmonellidae is very limited. We collected and isolated adult worms of three species in Japan that belong to the family Heligmonellidae, namely Heligmonoides speciosus (Konno, 1963) Durette-Desset, 1970 (Hs) from Apodemus argenteus, Orientostrongylus ezoensis Tada, 1975 (Oe) from Rattus norvegicus and Lagostrongylus leporis (Schulz, 1931) (Ll) from Pentalagus furnessi, and sequenced the entire internal transcribed spacer regions, ITS-1 and ITS-2 of ribosomal DNA. ITS-1 of Hs, Oe and Ll was 426, 468 and 449 bp in length, and had a G+C content of about 41, 41 and 37 %, respectively. ITS-2 of Hs, Oe and Ll was 297, 319 and 276 bp in length and had a G+C content of about 38, 40 and 28%, respectively. The data of Hs, Oe and Ll were compared with those of two other known species within the family Heligmonellidae, Calorinensis minutus (Dujardin, 1845) (Cm) and Nippostrogylus brasiliensis (Travassos, 1914) (Nb), and with those of two species of Heligmosomidae (Heligmosomoidea), Heligmosomoides polygyrus bakeri and Ohbayashinema erbaevae. Phylogenetic analysis placed Hs, Oe and Ll in the same clade with Cm and Nb, forming a Heligmonellidae branch in both ITS-1 and ITS-2, separate from the Heligmosomoidea branch. These results demonstrated that the ITS-1 and ITS-2 sequences are useful for differentiating the Heligmonellidae nematode species. This study is the first to describe the ITS-1 and ITS-2 sequences of Hs, Oe and Ll.