Calomys Waterhouse, 1837, is one of the most speciose genera of the Phyllotini tribe of the South American sigmodontine rodents. Distributed predominately in southern South America, the genus has been proposed to have originated in the central Andes with further differentiation as subsequent occupations of the lowlands of the continent occurred. In this study, 30 newly obtained sequences of the cytochrome-b gene from specimens collected in Brazil were analyzed in conjunction with data available in GenBank in an attempt to discern the dispersion patterns of this genus in the South American lowlands. The analyses support a scenario where a phyllotine lineage appeared in the Andes and later separated into 2 larger clades. Members of 1 clade remained in the highlands (C. musculinus, C. lepidus, and C. sorellus), experiencing some local differentiation. Members of the 2nd clade invaded the lowlands of South America, especially nonforested biomes, where they underwent intense differentiation resulting in species with wide distributions in the continent. In the lowland clade, the “callosus–venustus” group is more derived, is characterized by a larger body size, and has a broad distribution; differentiation of this group was probably accompanied by some reduction in chromosomal diploid numbers.