Three previously described Parasitodiplogaster spp., P. nymphanema, P. obtusinema and P. trigonema were re-isolated from their type locality, Barro Colorado Island Research Station, Smithsonian Tropical Research Institute, in Panama. The re-isolated materials were morphologically observed to compare with the original descriptions and molecularly characterised by DNA sequences of the near-full-length small subunit and D2-D3 expansion segments of the large subunit of the ribosomal RNA genes. Although the male tail characters, i.e., arrangement of genital papillae and spicule and gubernaculum morphologies, were close to the original descriptions, a compressed stoma with two (right subventral and dorsal) teeth was observed for the first time and confirmed in the newly re-isolated materials. The molecular phylogenetic analysis revealed that the three re-isolated species are close to P. laevigata, P. citrinema and P. popenema, forming the P. laevigata group in the genus, and this group was separated into three subgroups, P. citrinema + P. popenema (subgroup 1), P. nymphanema + P. obtusinema (subgroup 2) and P. laevigata + P. trigonema and three undescribed species (subgroup 3). The P. laevigata group is characterised by a relatively compressed stoma with two (right subventral and dorsal) teeth, arrangement of genital papillae (three or four precloacal and four postcloacal pairs), but distinguishable from each other by stomatal morphology, i.e., the shortest and most compressed being in subgroup 3, widest in subgroup 1, and intermediate in subgroup 2. Furthermore, a large and long spicule and gubernaculum were observed in subgroup 2 as its hypothesised apomorphy.