Rodents of the subfamily Sigmodontinae comprise a highly diversified group in the Atlantic Forest, with semifossorial, terrestrial, semiaquatic, scansorial, and arboreal forms. In this study, we analyzed morphometric variation in humerus, scapula, ulna, radius, femur, tibia, and pelvis to investigate its possible relationship with the different types of locomotion recorded in the literature. Skeletal characters were measured in 321 specimens belonging to 29 species and 19 genera either restricted to or recorded in this ecoregion. Multivariate morphometric analyses (principal component and canonical variate analyses) arranged individuals of different genera in groups congruent with the different types of locomotion. This arrangement was more clearly defined when analyses included only forelimb measurements, indicating that most of the variation in appendicular traits associated with the different locomotor modes occurs in the forelimb skeleton. Semifossorial forms exhibited the most distinct appendicular morphology, as well as the greatest frequency of endemism among analyzed species. These results suggest that this mode of locomotion led to greater differentiation in semifossorial Atlantic forest sigmodontines than in terrestrial and arboreal forms, which were found to have more subtle differentiation and fewer endemics. Scansorial species could not be set apart from terrestrial ones in terms of appendicular morphology, suggesting that these two modes of locomotion are the most similar and generalized for the group, as they occur in most lineages in the subfamily. The results of this study corroborate previous observations on the relevance of appendicular characters in the differentiation of species and genera in the subfamily Sigmodontinae. J. Morphol. 2013. © 2013 Wiley Periodicals, Inc.