The forces acting on the vertebral column of varanid lizards differ greatly during swimming and walking. To examine the long-term impact of these forces, the dorsal vertebrae of terrestrial and aquatic species of Varanus were compared using 3-D laser scanning and morphometric analysis. There were significant differences between the two groups in vertebral anatomical features, particularly in the articular surfaces. Further analysis demonstrated that the dorsal vertebrae could be significantly divided into three groups of nearly equal size: the sternal group (dorsal vertebrae 1–7), the middle group (dorsal vertebrae 8–14), and the pelvic group (dorsal vertebrae 15–22). Within each of these groups there was significant differences between the terrestrial and aquatic species; these differences were least in the more conserved sternal region and greatest in the highly variable pelvic region. The results suggest that vertebral morphometrics can be used as a tool to further delineate the habitat preferences of extinct varanoid lizards, and could serve to highlight the functional transitions between terrestrial and aquatic species.