Congruence between patterns of localized, hierarchical variation in cranial shape and topological, molecular phylogenetic structure was investigated in a monophyletic lineage of Neotropical spiny rats of the genus Trinomys. Levels of organizational complexity in shape were assessed from two-dimensional coordinates of anatomical landmarks for dorsal, ventral and lateral views of the cranium, and the scale of variation in cranial shape was decomposed hierarchically using the statistical formalism of geometric morphometrics. The patterns of variation in cranial shape were evaluated for the five taxa of Trinomys in terms of ordinations in the reduced space of relative warps, with the scores of partial warps weighted to emphasize the hierarchical localization of shape differences in different geometric scales. The fit of the morphological shape data to the molecular phylogeny and analysis of the correlation between measures of the differences in shape and molecular phylogenetic distances demonstrated that only variation in small, localized scales in cranial shape in the lateral view of the cranium was congruent with molecular phylogenetic structure. The significance and perspectives of the application of geometric descriptors of shape and the identification of scales of variation for the study of morphological and molecular evolution are discussed. © 2003 The Linnean Society of London, Biological Journal of the Linnean Society, 2003, 80, 385–396.