Overwater dispersal and subsequent allopatric speciation contribute importantly to the species diversity of West Indian Anolis lizards and many other island radiations. Here we use molecular phylogenetic analyses to assess the contribution of overwater dispersal to diversification of the Anolis carolinensis subgroup, a clade comprising nine canopy-dwelling species distributed across the northern Caribbean. Although this clade includes some of the most successful dispersers and colonists in the anole radiation, the taxonomic status and origin of many endemic populations have been ambiguous. New mitochondrial and nuclear DNA sequences from four species occurring on small islands or island banks (Anolis brunneus, Anolis longiceps, Anolis maynardi, Anolis smaragdinus) and one species from the continental United States (A. carolinensis) are presented and analysed with homologous sequences sampled from related species on Cuba (Anolis allisoni and Anolis porcatus). Our analyses confirm that all five non-Cuban species included in our study represent distinct, independently evolving lineages that warrant continued species recognition. Moreover, our results support Ernest Williams's hypothesis that all of these species originated by overseas colonization from Cuban source populations. However, contrary to Williams's hypothesis of Pleistocene dispersal, most colonization events leading to speciation apparently occurred earlier, in the late Miocene–Pliocene. These patterns suggest that overwater dispersal among geologically distinct islands and island banks is relatively infrequent in anoles and has contributed to allopatric speciation. Finally, our results suggest that large Greater Antillean islands serve as centres of origin for regional species diversity.