Abstract Abiotic factors can influence genetic and phenotypic divergence in several ways, and identifying the mechanisms responsible for generating this variation is challenging. However, when evaluated in combination, ecological characteristics and genetic and phenotypic information can help us to understand how habitat preferences can influence morphological and genetic patterns exhibited by taxa distributed between distinct biomes, such as the Atlantic Forest and Pampas biomes in South America. By combining distributional, environmental, phenotypic and genomic information from a habitat-specialist semi-aquatic rodent (Holochilus brasiliensis), we quantified the relationship between ecological niche differences and the phenotypic and genetic variation. The results demonstrate notable segregation among the ecological niches of H. brasiliensis within each biome, although we could not refute the hypothesis of niche similarity or equivalency. Such differences are consistent with a solid morphometric variation associated with the size of these rodents. However, the ecological and morphometric differentiation is not accompanied by the same pattern of genetic variation. Despite differences in the connectivity patterns in both biomes, the genetic differences corroborate a consistent level of migration history between biomes. Additionally, the association tests show that the environment explains a small and non- significant part of the genetic variation but a significant portion of the morphometric variation.