Xylopia emarginata Mart., a tree species that grows in the riparian forests, is characterized by is tolerance to water-saturated soils, which enables its use for restoration of riparian ecosystems. The objective of this study was to determine the genetic structure, levels of genetic variability and gene flow in two natural populations divided naturally in five sub-populations, with altitude ranging from 940 to 1010 m. The populations are located at Itumirim (S 21°21'42,8''; W 44°46'0,52''), in Southern Minas Gerais state. Samples of leaf tissue were taken from adult plants from each population for isoenzyme electrophoresis analysis. The genetic equity in sub-population 1 was 36%; in 2 was 9%, in 3 was 27%; and in 4 and 5 was 72%. These values indicate that the sub-populations 1, 2 and 3 can be more subject to genetic drift. The observed heterozygosity (Ĥo) was 0.484 in the A population and 0.722 in the B population. The expected heterozygosity (Ĥe) was 0.395 and 0.425 in the A and B populations respectively. The estimated parameters revealed no inbreeding in the populations (F̂ = -0.29). The low gene flow estimated among sub-populations could be due the presence of a mountain ridge, which may be acting like a geographic barrier. This result was confirmed by the high genetic identity between the sub-populations 1 and 4. The sub-populations 1, 4 and 5 are closer genetically, even being more distant geographically. These data are important to seed collection programs aiming genetic conservation.