Morphological variation in size and shape of organisms has physiological, ecological, and evolutionary relevance. In this context, an important step in the identification of evolutionary units is to identify groups of populations occupying a continuous geographic space, at both genetic and morphological levels. The freshwater turtle Hydromedusa is endemic to the Neotropical region and inhabits water bodies of the Paraná–La Plata basin of Argentina and coastal streams of Brazil and Uruguay. The genus is present in the fossil record since the Paleocene (56 mya) and currently consists of only 2 extant species. Hydromedusa tectifera has the widest distribution, from Santiago del Estero in Argentina to the State of Sao Paulo in Brazil. The goal of this work was to study the patterns of morphological variation of H. tectifera through most of its geographical range. Herein, we report morphological variation of carapace shape in this species associated with developmental and historical parameters. Our results support the hypothesis that at least part of the morphological variation found is associated with population variation among basins, possibly as a result of reduced gene flow among their populations. This variation is shown in both linear and geometric morphometry analyses. Sea level fluctuations that occurred in the region during the last 15 million years could have caused the current differentiation.