A unique characteristic of mammals is a vertebral column with anatomically distinct regions, but when and how this trait evolved remains unknown. Here we reconstruct vertebral regions and their morphological disparity in the extinct forerunners of mammals, the non-mammalian synapsids, to elucidate the evolution of mammalian axial differentiation. Mapping patterns of regionalization and disparity (heterogeneity) across amniotes reveals that both traits increased during synapsid evolution. However, the onset of regionalization predates increased heterogeneity. Based on inferred homology patterns, we propose a “pectoral-first” hypothesis for region acquisition. Evolutionary shifts in forelimb function in non-mammalian therapsids drove increasing vertebral modularity prior to differentiation of the vertebral column for specialized functions in mammals.