Prunus mira, an important economic fruit crop and ancestral peach species, is widely distributed along the Yarlung Zangbo Grand Canyon and tributary basins in the Tibetan plateau. Due to infrastructure construction and deforestation, natural habitat of P. mira has been severely damaged, leading to reduction in its distribution range and population size and it is listed as a critically endangered species in China’s list of National Key Protected Wild Plants. In this study, 85 novel single nucleotide polymorphism (SNP) markers were developed based on restriction-site associated DNA sequencing. The minor allele frequency, observed heterozygosity, and expected heterozygosity ranged from 0.064 to 0.748, 0.086 to 0.846, and 0.126 to 0.562, respectively. The PIC ranged from 0.117 to 0.423. Ten loci showed significant deviations from the Hardy–Weinberg equilibrium after Bonferroni correction (p < 0.05). These novel SNPs can be useful for future population and conservation genetics studies on this species.