Phylogenetic relationships of 11 species of cotton rats (Genus Sigmodon) were assessed using nucleotide variation in the mitochondrial cytochrome-b gene. Results of the phylogenetic analyses do not support the conventional composition of the Sigmodon hispidus and S. fulviventer species groups. Instead, 3 species groups are recognized: hispidus (S. alleni, S. arizonae, S. hirsutus, S. hispidus, S. mascotensis, S. ochrognathus, and S. toltecus), fulviventer (S. leucotis, S. fulviventer, and S. peruanus), and alstoni (S. alstoni). Sequence divergence values ranged from 8.7% to 21.2%, indicating that divergence events within the genus occurred approximately 2.5–5.6 × 10⁶ years ago. Taxa comprising the alstoni and fulviventer groups appear to have evolved early, whereas members of the hispidus group evolved more recently. Molecular data reflect an early origin (7 × 10⁶ years ago) and support the hypothesis that cotton rats may occupy a basal position among South American sigmodontines.