Trinomys is a very diverse mammalian genus of eastern Brazil, comprising ten species. Eight of them have remained in their original biome, the Atlantic Forest, while two others occupy the semi-arid Caatinga. One of the species from the Atlantic Forest, T. eliasi, also inhabits adjacent restingas, an open vegetation ecosystem on sandy sediments of marine origin. Here we assess the impact of colonization of very different environments on the rates of phenotypic change in Trinomys. Multivariate analyses showed that species that had remained in the Atlantic Forest maintained a strong cohesion in skull shape and size. By contrast, colonization of the Caatinga was associated with remarkable differentiation and a strong increase in the rates of phenotypic change, 7–17 times higher than in the Atlantic Forest. Trinomys eliasi from restinga differed from forest samples and other species by a recent change in morphology. Our findings showed that occupation of remarkably different ecosystems had a significant impact in the phenotypic evolution of Trinomys.