Studies of forest fragmentation effects upon the genetic structure of selected species are important for planning genetic conservation strategies. They can indicate the genetic behavior of species with similar ecological characteristics. Esenbeckia leiocarpa is a myophilic and autochoric Rutaceae tropical tree species occurring in an aggregated distribution in Tropical Moist Seazonal Brazilian Atlantic Forest, with several subpopulations constituting a forest fragment population. Leaf tissues of individuals from two subpopulation from a 2178 hectares forest fragment and two subpopulations from a 76 hectares forest fragment were analysed in alozymes horizontal electrophoresis in corn starch gel support. A1 the loci showing from 1 to 4 alleles. The loci Pgm-1, Idh-1 and Mdh-2 were monomorphic. Skdh-1, 6Pgdh-2, Mdh-1, Mdh-3, Mdh-4, Est-1, Prx-1 and Pgi2 loci were polymorphic. The average of observed heterozigosity (Ho) and expected heterozigosity (Ĥe) were not statistically signifficant. The f̂ and F̂ values, suggested that the populations are in Hardy-Weinberg equilibrium. The genetic diversity among populations (θ̂p) was 12.1% for adult individuals and 8.7% for the progenies showing diferences among populations. The allele frequencies of subpopulations from the larger fragment showed greater differences than those from the smaller fragment. Allele fixation levels were more homogenous in the smaller fragment subpopulation. The gene flow was the hight among subpopulations in Ibicatu fragment. Hierarquical analysis of genetic distribution within and among populations and subpopulations showed that the major part of genetic variability of species is maintained within subpopulations. 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