Population genetic diversity contributes to the protection and utilization of germplasm resources, especially via genetic breeding. In the present study, start codon targeted polymorphism (SCoT) molecular markers were used to study the genetic diversity of 278 individuals from 10 Prunus sibirica populations in Inner Mongolia. A total of 289 polymorphic bands were amplified with 23 SCoT primers, showing a polymorphism percentage of 97.94% and an average of 12.6 polymorphic bands per primer. The SCoT21, SCoT32, and SCoT53 primers amplified up to 17 bands, and the polymorphism percentage was 100%. The minimum number of bands amplified by SCoT3 was 9, and the polymorphism percentage was 90%. Therefore, SCoT molecular markers were shown to be highly polymorphic and suitable for genetic diversity studies of Prunus sibirica in Inner Mongolia. The analysis of molecular variance (AMOVA) showed that 39% of the observed genetic differentiation occurred among populations and 61% occurred within populations, indicating that the genetic differentiation within populations was greater than that among populations. The results of the unweighted pair-group method with an arithmetic (UPGMA) cluster analysis, principal coordinate analysis (PCoA) and STRUCTURE analysis were basically the same and divided the 278 individuals from the 10 populations into 2 groups. The results indicated that the efficient SCoT molecular marker-based genetic diversity analysis of Prunus sibirica in Inner Mongolia can provide a reference for Prunus sibirica variety breeding and resource development.