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Calophyllum brasiliense Camb. (Clusiaceae) is a tree of great ecological plasticity and it is present in different forest physiognomy. Due to its preference to water-saturated soils, it is considered specialist in habitat, due to this, it occurs frequently in the riparian forest. In order to access and understand the inter and intrapopulation genetic variability patterns, two populations of C. brasiliense in gallery forest were sampled. The results obtained by isoenzyme electrophoresis analysis showed a high heterozygosity for this species, Ĥ0equal 0.444 and 0.492. The genetic structure analysis indicated the absence of intra and inter populations inbreeding ( f = -0.078; F = -0.063). Most of the genetic variability was distributed within the populations (θ = 0.14) and the gene flow was low ( Nm = 0.83). The coancestry coefficient estimated showed positive spatial structure in small distance classes. This information is important to programs of conservation genetics in situ and ex situ of the species. Moreover, it is necessary to improve the preservation and the maintenance of natural populations of C. brasiliense, since this species demonstrates fragility to factors that put at risk its genetic variability.