Guaçatonga (Casearia sylvestris) is a native plant of the Atlantic Forest, with high medicinal potential and relevance for reforestation programs. The aim of this study was to characterize, with microsatellite markers, two populations of C. sylvestris from remaining areas of the Atlantic Forest in the State of São Paulo. High allelic variation was found in both populations (NA = 101 and 117; AR = 12.5 and 14.4), although with high endogamy coefficients (f = 0.640 and 0.363). Estimates of genetic structure suggested the presence of considerable genetic divergence between the populations (FST = 0.103); however, there was no spatial genetic structure within the populations. Genetic divergence may have occurred due to decreased gene flow between the fragmented populations as the result of deforestation. The results of this study demonstrate the importance of genetic diversity and its characterization in native plants within remaining forest areas for the management and restoration of such areas.