Understanding the genetic diversity and differentiation of the genetic resources of a species is important for the effective use and protection of forest tree resources. Ex situ development is a common method for the protection of genetic diversity and an essential resource for users who require ready access to a species’ germplasm. In this study, we collected seeds of black locust (Robinia pseudoacacia L.) from 19 provenances, covering most of its natural distribution; we randomly selected 367 tender leaves with well-grown and different maternal strains from this group for further analysis. Forty-eight simple sequence repeat (SSR) primers were successfully selected from 91 pairs of SSR primers using native-deformation polyacrylamide gel electrophoresis. In addition, we identified identical genotypes among all individuals and evaluated the quality of the markers. From this, 35 loci were confirmed for analyses of genetic diversity and differentiation of the black locust provenances, which contained 28 expressed sequence tag-derived simple sequence repeats (EST-SSRs) and 7 genomic DNA-derived simple sequence repeats (G-SSRs). We observed high genetic diversity among the native black locust provenances, from which Wright’s fixation index and molecular variance suggested that a majority of the genetic differentiation variation could be attributed to within-provenance differences. The genetic distance and identity results indicated that geographic distance was not a dominating factor influencing the distribution of black locust. This is the first study to evaluate provenance genetic variation in native black locust samples using two types of SSR markers, which provides a comprehensive theoretical basis for ex situ conservation and utilization of genetic resources, with an emphasis on breeding applications.