Anadenanthera colubrina (Acol) and Anadenanthera peregrina (Aper) (Fabaceae) are two species popularly known as “angicos” that occur in seasonally dry tropical forest (SDTR) remnants in Brazil. Since many of the morphological characteristics of Anadenanthera species are superimposed and species-specific characteristics are difficult to observe, their identification is complex. Therefore, in this research, a set of ISSR (Inter-Simple Sequence Repeat Polymorphic DNA) molecular markers was standardized, aiming to characterize A. colubrina and A. peregrina species and study the genetic diversity of three populations of each species located within a fragmented landscape in São Paulo State, southeastern Brazil. Seven ISSR markers (UBC 2, 820, 851, 858, 864, 866, and 886) that show polymorphism for both species were used. The Bayesian cluster, PCoA and dendrogram analysis show that the total sample divides into two groups corresponding to each species. Also, a genetic divergence (Gst = 0.143) and a high number of migrants per generation (Nm = 3.0) were detected between them. The Acol populations showed significantly higher values for mean genetic diversity (h = 0.30) than Aper (h = 0.25) (p < 0.05). The ISSR marker UBC2250bp showed species-specific electrophoretic fingerprints for both species. The molecular tools generated herein support the conservation of Anadenanthera sp. and the restoration of vegetation where the species naturally occurs.