The relative influence of Neogene geomorphological events and Quaternary climatic changes as causal mechanisms on Neotropical diversification remains largely speculative, as most divergence timing inferences are based on a single locus and have limited taxonomic or geographic sampling. To investigate these influences, we use a multilocus (two mitochondrial and 11 nuclear genes) range-wide sampling of Phyllopezus pollicaris, a gecko complex widely distributed across the poorly studied South American ‘dry diagonal’ biomes. Our approach couples traditional and model-based phylogeography with geospatial methods, and demonstrates Miocene diversification and limited influence of Pleistocene climatic fluctuations on P. pollicaris. Phylogeographic structure and distribution models highlight that persistence across multiple isolated regions shaped the diversification of this species complex. Approximate Bayesian computation supports hypotheses of allopatric and ecological/sympatric speciation between lineages that largely coincide with genetic clusters associated with Chaco, Cerrado, and Caatinga, standing for complex diversification between the ‘dry diagonal’ biomes. We recover extremely high genetic diversity and suggest that eight well-supported clades may be valid species, with direct implications for taxonomy and conservation assessments. These patterns exemplify how low-vagility species complexes, characterized by strong genetic structure and pre-Pleistocene divergence histories, represent ideal radiations to investigate broad biogeographic histories of associated biomes.