AbstractCruzia tentaculatais a helminth parasite of marsupials and has a wide geographic distribution from Mexico to Argentina. The aim of this study was to analyse the genetic population structure of this nematode along the Atlantic Forest biome.Cruzia tentaculataspecimens were recovered fromDidelphis aurita,Didelphis albiventrisandPhilander quicain 9 localities. Morphological and morphometric data were investigated for phenotypic diversity among localities and hosts using multivariate discriminant analysis of principal components. Phylogenetic relationships ofC. tentaculatawere determined using maximum likelihood and Bayesian inference. The population structure was analysed by fixation indices, molecular variance analysis, Tajima'sDand Fu'sFs neutrality tests, Mantel tests and Bayesian clustering analysis. A higher significant morphometric difference for males was observed between localities. In the haplogroup networks, 2 groups were recovered, separating locations from the north and from the south/southeast. The morphometric variation inC.tentaculatabetween different localities was compatible with this north and southeast/south pattern, suggesting adaptation to different ecological conditions. Population genetic analyses suggested a pattern of evolutionary processes driven by Pleistocene glacial refugia in the northeast and southeast of the Atlantic Forest based on the distribution of genetic diversity.