In this study, the genetic diversity and differentiation of 10 natural Prunus pseudocerasus Lindl. populations were investigated using inter-simple sequence repeat (ISSR) markers. Totally, 18 selected primers generated 150 loci, with an average of 8.33 bands per primer. The results showed that the percentage of polymorphic bands (PPB) was pretty low at the population level (PPB = 1.13–32%), but relatively high at the species level (PPB = 84%). Besides, a high level of genetic differentiation among populations was detected based on the gene differentiation coefficient (GST = 0.7118) and the hierarchical analysis of molecular variance (AMOVA) (ΦST = 64.53%, P < 0.001), in line with the low inter-population gene flow (Nm = 0.2025). Moreover, Mantel test revealed a significant correlation between genetic and geographic distances among the populations (r = 0.5272, P < 0.005). The high level of intraspecific genetic diversity was probably related with its life history traits, while its small population size and the resultant high levels of genetic drift and inbreeding might explain the low genetic diversity within populations. The relatively high inter-population genetic differentiation was largely attributed to its small population size, habitat fragmentation, the mode of pollen and seed dispersal, and geographic isolation. Based on the present study, conservation strategies were proposed to preserve this valuable natural germplasm resource.