Abstract Aplectana membranosa is a cosmocercid nematode that shows affinity with various amphibian and reptile hosts, being considered a generalist species. To date, no studies have investigated the influence of host and locality in the morphological variation of this species. Thus, we analysed morphological and morphometric characters of 260 specimens of A. membranosa collected from 9 host species and 7 different localities. To complement the metric studies, we conducted phylogenetic analyses using the ribosomal genes 28S and internal transcript spacer 1 ( ITS1 ) to determine the phylogenetic position of the species and its divergence. In the present study, it was possible to observe the cloacal papillae pattern of the species through scanning electron microscopy, and we found no morphological variation in the specimens of A. membranosa from various hosts in different localities in Brazil. The study showed low variation in all data. However, despite the low variation, we found that external environmental conditions, such as climate and latitude, influence its variation. Molecular analyses highlighted that the separation of Cosmocercidae members may be related to geographic distribution and population genetic divergence. Thus, the results illustrated in this study reiterate the importance of using integrative data to better elucidate the family’s taxonomic and evolutionary history.