Genetic diversity and population structure analysis were carried out on the newly collected germplasm resources of cultivated species Malus prunifolia of Malus Mill. by use of the fluorescent SSR molecular markers. The identification of the genetic diversity and structure in and among populations can provided references for the collection and preservation of germplasm resources of Malus, and for the selection of rootstock breeding. Fingerprint data were constructed using fluorescent SSR molecular markers, genetic diversity was analyzed using GenAlEx 6.501, Neighbour-Joining tree was constructed using POPULATION 1.2, and population structure was analyzed using STRUCTURE 2.3.4. The results showed that 390 polymorphic alleles were detected by 19 pairs of SSR primers, the average allele number was 20.526, and the average effective allele number was 9.399. The average values of heterozygosity and expected heterozygosity were 0.706 and 0.868, respectively. The Shannon index was 2.446. The genetic diversity of Malus prunifolia in this study was higher than the other species of Malus Mill. before. The cluster analysis based on Nei genetic distance can be divided into three groups for 155 accessions at 0.9167. Three groups are close with each other, and most groups are not related to geographical location. The population structure analysis of all accessions was divided into two groups. Clustering results coincide with the population structure. Q value of 150 accessions was higher than 0.6, and these accessions had a relatively single blood relationship.