Studies of genetic diversity in natural populations are important for the definition of conservation strategies, especially in populations reduced by processes of fragmentation and continuous forest extraction. Molecular markers stand out as interesting tools for these studies. The objective of this research was to characterize the diversity and genetic structure of Plathymenia reticulata (Fabaceae), occurring in two fragments of the Montana Semideciduous Forest in the southern of Espírito Santo State, Brazil, using inter-simple sequence repeat (ISSR) molecular markers. DNA samples from 149 individuals were analyzed using 10 ISSR primers, generating 156 fragments, of which 101 were polymorphic (64.74%). The individuals sampled were classified into three units: adult trees (A), a mixture of progenies (B), and young regenerating individuals (C). The number of loci used (N = 101) was greater than that established as optimal number (N = 88), indicating precision in analyses. The genetic diversity index of Nei (H’ = 0.407) and the Shannon index (I = 0.594) were found to have high genetic diversity. Besides, through the diversity parameters evaluated, it was possible to confirm that in the areas of natural regeneration and progeny mix there is genetic diversity equivalent to that found in adults. The analysis of molecular variance indicated that most of the genetic variation is found within the groups (96.53%). Genetic differentiation among adult trees was low (ΦST = 0.03) indicating that high gene flow rates (Nm = 12.70) are counteracting the effects of genetic drift. The data obtained allowed to evaluate the potential of adult trees as matrices for seed collection and to obtain seedlings with confirmed genetic variability.