The Galápagos archipelago is characterized by a high degree of endemism across many taxa, linked to the archipelago's oceanic origin and distance from other colonizing land masses. A population of ~500 American Flamingos (Phoenicopterus ruber) resides in Galápagos, which is thought to share an historical origin with the American Flamingo currently found in the Caribbean region. Genetic and phenotypic parameters in American Flamingos from Galápagos and from the Caribbean were investigated. Microsatellite and mitochondrial DNA markers data showed that the American Flamingo population in Galápagos differs genetically from that in the Caribbean. American Flamingos in Galápagos form a clade that differs by a single common nucleotide substitution from American Flamingos in the Caribbean. The genetic differentiation is also evident from nuclear DNA in that microsatellite data reveal a number of private alleles for the American Flamingo in Galápagos. Analysis of skeletal measurements showed that American Flamingos in Galápagos are smaller than those in the Caribbean primarily due to shorter tarsus length, and exhibit differences in body shape sexual dimorphism. American Flamingo eggs from Galápagos have smaller linear dimensions and volumes than those from the Caribbean. These findings are consistent with reproductive isolation of the American Flamingo population in Galápagos.