We assessed the genetic and morphological differences between the two largest breeding colonies of Audouin's Gull Larus audouinii, an endemic seabird species of the Mediterranean region. The two colonies comprise c. 75% of the total world population and are 655 km apart. The Ebro Delta colony was formed recently and, after dramatic growth mainly due to high rates of both immigration and reproductive success, is now the largest in the world (more than 60% of the total population). The Chafarinas Islands support an ancient colony with relatively little fluctuation in breeding numbers. The two colonies also differ greatly in environmental conditions, with the Ebro Delta being a higher quality breeding site. Very little movement occurs between the two colonies. We collected morphological data and blood samples from both colonies. Polymorphic microsatellite markers were used to study the genetic differentiation. These showed no significant variation between colonies, nor evidence of a founder effect in the Ebro Delta. Individuals from the Ebro Delta were larger than those from Chafarinas, the difference being greater for males. This probably reflects a stronger male susceptibility to worse environmental conditions during chick growth at the Chafarinas Islands.