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Aim Pilosocereus is one of the richest and most widespread genera of columnar cacti, extending from south-west USA to southern Brazil. Most species occur in the seasonally dry tropical forest biome but can also be found in xeric microhabitats inside woody savannas (Cerrado) and moist forests (Brazilian Atlantic forest). The genus exhibits a highly disjunct distribution across the Neotropics. Using a 90% complete species-level phylogeny, we reconstructed the spatio-temporal evolution of Pilosocereus to explore the historical factors behind the species richness of Neotropical dry formations. Location South America, Mesoamerica, Caribbean, south-western North America. Taxon Genus Pilosocereus (Cactaceae, Cactoideae, Cereeae). Methods We used plastid and nuclear DNA sequences and Bayesian inference to estimate phylogenetic relationships and lineage divergence times. Ancestral ranges were inferred within the Pilosocereus subgenus Pilosocereus s. s. clade using the Dispersal-Extinction-Cladogenesis model in a Bayesian framework to account for parameter estimation uncertainty and the effect of geographical distance on dispersal rates. Results Pilosocereus was recovered as polyphyletic, with representatives of other Cereeae nested within. The Pilosocereus subgenus Pilosocereus s. s. clade originated around the Pliocene-Pleistocene transition (2.7 Ma), probably within the Caatinga seasonally dry tropical forest (SDTF) formation. Species divergences were dated in the Middle and Upper Pleistocene, often constrained to the same geographic region but also associated to migration events to other xeric habitats in Mesoamerica and northern South America; dispersal rates were not dependent on distance. Main conclusions Diversification dynamics in the Pilosocereus subgenus Pilosocereus s. s. clade agree with other infrageneric studies in cacti. Species divergence was rapid, driven by in situ diversification and migration events between SDTF dry formations and xeric microhabitats within other biomes and probably linked to Pleistocene climatic changes. This dynamic history differs from that found in woody SDTF lineages, which are older in age and characterized by low-dispersal rates and long-term isolation.