Aplectana hylambatis (Baylis, 1927) is a widespread nematode in South America that infects different species of anurans from different families. Although the morphology of this species shows intraspecific variations and is well studied, A. hylambatis does not have genetic characterisation. Therefore, the present study aimed to provide the first genetic characterisation of this parasite, based on 18S and 28S rDNA sequences, as well as to evaluate its phylogenetic position in the family Cosmocercidae. The present specimens showed no major morphological variations and were assigned to A. hylambatis mainly based on the shape of spicules and the presence of mamelon-like structures slightly anterior to the vulva. The genetic characterisation confirmed the close relationship between A. hylambatis and other cosmocercids, and the 28S sequences were more informative for phylogenetic reconstruction, especially for those relationships at higher and intermediate levels. Aplectana hylmabatis was in a basal position within a clade formed by other congeners and species of Cosmocerca. The geographic origin may have influence in the divergence process of Aplectana spp. and genus was non-monophyletic and closely related to Cosmocerca, similar to previous findings. The present results will be useful for future studies on the phylogeny of Cosmocercidae, since several genera and species in this family still lack genetic characterisation. Furthermore, the 28S sequences can be useful in genetic studies of A. hylambatis populations, since this species appears to have phenotypic variations.