Roseate Spoonbill (Platalea ajaja) feather samples were collected from nestlings in breeding colonies in the Birama Swamp (n = 34 individuals) and Sabana-Camagüey Archipelago (n = 38 individuals) of Cuba. Genetic diversity and structure were determined from nestling feather samples using five microsatellite loci, and sibling relationships and mating system were inferred. The number of alleles per locus ranged from two to eight, with an average of 5.6 alleles per locus and a total of 28 alleles, and no genetic structure was found between the Cuban colonies. Sibship determined from 52 dyads (nestling-pairs) showed deviation from genetic monogamy, with seven of 24 nests presenting evidence of extra-pair paternity (EPP) and five nests presenting evidence of conspecific brood parasitism (CBP). Genetic diversity was similar to that reported for colonies from the United States and Brazil, and EPP was more frequent than CBP as an alternative to monogamy.