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We examined mitochondrial cytochrome b sequence variation in masked Sula dactylatra, red-footed S. sula, and brown S. leucogaster boobies sampled from islands in the central and eastern Pacific Ocean and in the Caribbean Sea. Each species showed a different phylogeographic pattern. Whereas haplotypes in masked and red-footed boobies were shared across the central and eastern Pacific (i.e., across the Eastern Pacific Basin), brown booby haplotypes were not shared across the Eastern Pacific Basin. Although most masked booby haplotypes from the Pacific were distinct from those in the Caribbean, one haplotype was shared across the Isthmus of Panama. Red-footed and brown boobies, however, did not share haplotypes across the Isthmus of Panama. We estimate that divergence of these regional populations occurred within the last 560,000 years. Thus, the Isthmus of Panama and the Eastern Pacific Basin (albeit to a lesser degree) appear to have played a role in the diversification of these species.