The Water Opossum, Chironectes minimus (Didelphimorphia, Didelphidae), is the only semi-aquatic marsupial and can be easily identified for its uniquely patterned water resistant pelage and webbed hind-feet. The species is distributed along the Neotropical region (from Southern Mexico to Northwestern Argentina) and is currently divided on four subspecies (C. m. argyrodytes, C. m. panamensis, C. m. minimus, C. m. paraguensis), based on its geographical distribution. We evaluated the geographic variation in Chironectes minimus, using traditional and geometric morphometrics techniques to capture the size and shape of the skull to identify differences between the populations that could relate to existing subpsecies or populations that would merit particular taxonomic attention. We took 13 skull measurements, and digitized 37, 18, and 23 2D landmarks on the dorsal and lateral view of the cranium and on the lateral view of the mandible, respectively. The sample consisted on 135 individuals from 66 localities. Analyses of both datasets showed that the Southern South America and Northern Central America animals differ from the other populations. Geometric morphometrics shows on the three views that the Amazon animals differ from the others probably due to existing geographical barriers. These morphological differences among populations of the Water Opossum support the need for a proper phylogenetic assessment of these populations, in order to clarify their taxonomic status.