The Rufous-tailed Hummingbird (Amazilia tzacatl) is a common resident of the neotropical lowlands of Middle America and northern South America. The Escudo Hummingbird (A. t. handleyi), the most distinctive of the five subspecies of A. tzacatl, is endemic to Isla Escudo in Caribbean western Panama. Mitochondrial DNA (mtDNA) sequence variation from across most of the species' range showed five well-defined but shallow clades (maximum uncorrected distance 2.4%) that only partially agreed with subspecific taxonomy. A widespread Middle American clade ranges from southeastern Mexico to central Caribbean Panama and includes birds collected on the Bocas del Toro archipelago. The Escudo Hummingbird fell within this clade and was slightly differentiated (two unique substitutions; uncorrected distance ∼0.2–0.5%). Two additional clades occur in the Pacific regions of southern Middle America. A fourth clade is endemic to eastern Panama (eastern Panamá and Darién provinces); a fifth is found in northwestern South America and in Darién. Secondary contact between clades occurs at three sites: between clades I and III in northwestern Costa Rica, between clades I and II in western Panama, and between clades IV and V near the Panama-Colombia border. The last case is likely due to recent expansion into the region from two directions. Thus the history of Amazilia tzacatl demonstrates a tendency for the formation of monophyletic mtDNA clades, likely as the result of geographic isolation, but also a propensity for secondary contact of these clades, a phenomenon recovered in many other phylogeographic studies of neotropical birds.