Metrodorea nigra A. St.-Hil. (Rutaceae) is a submontane tropical tree species that develops preferably in the shade and innermost preserved regions of the semi-deciduous forests of Brazil; as a result, its presence can indicate good levels of conservation. Therefore, it is important to have a set of molecular markers to facilitate genetic studies on this species of tree. The goal of this study was to perform genetic analyses of three generations of one population of M. nigra located in an urban landscape (Ribeirão Preto, São Paulo state) using a new set of microsatellite molecular markers (i.e., SSR—simple sequence repeat). A genomic library was developed from M. nigra, and 11 putative molecular markers were isolated and characterized using samples from different life stages. Seven SSR markers amplified and showed polymorphism across 61 M. nigra samples. A total of 83 alleles were detected, with an average of 11.85 alleles per locus. The genetic diversity index (He) across M. nigra generations was high and ranged from a mean of 0.804 (saplings) to 0.827 (adults). These results show the resilience of M. nigra population located in Bosque Municipal de Ribeirão Preto-SP, Brazil, to adverse effects of fragmentation, and can serve as a base for conservation programs. Furthermore, more than 50 % of the primers were also amplified for the congeneric tree species Metrodorea stipularis. Overall, the new marker set developed here will be useful for studies of population genetics of M. nigra and other key tree species from the Atlantic Rainforest of Brazil.