Five breeding colonies of the Roseate spoonbill (Aves: Platalea ajaja) from two Brazilian wetland areas (Pantanal and Taim marshes) were sampled, and domain I of the mitochondrial DNA control region (483 bp) was sequenced in 50 birds. The average haplotype diversity (h = 0.75, s = 0.071) and average nucleotide diversity (pi = 0.004, s = 0.003) were evaluated, and nonsignificant differences were found among the colonies studied. The lack of differentiation among breeding colonies revealed by AMOVA analysis was explained either as a consequence of high gene flow or recent expansion. The significantly negative results of the neutrality tests (Fu's F ( s ) = -23.271, P < 0.01; Tajima's D = -1.941, P < 0.01) associated with the star shape of the haplotype tree and mismatch distribution data are evidence supporting the idea that these populations underwent a recent demographic expansion in the Pantanal region. The average time since the expansion is estimated to be 25,773 years, and this agrees with a period of increased moisture that occurred during the last glacial period.