Korla fragrant pear is a high-quality local pear variety native to Xinjiang, China. Currently, the low fruit-setting rate and low calyx shedding rate problems in Korla fragrant pears have been highlighted, which seriously affect the fruit yield and quality. It is of great significance to research the fruiting characteristics and molecular-assisted identification of Korla fragrant pear bud mutation materials for enriching the germplasm resources of Korla fragrant pear. In this research, a natural pollination group (YB) of Korla fragrant pear bud mutation materials and a natural pollination group (CK) of Korla fragrant pears were established. On the fruiting characteristics, the fruit-setting rate and calyx-removal rate of the two groups were investigated. In terms of fruit quality, the fruit shape index, fruit specific gravity, soluble solids content, sugar:acid ratio, soluble sugar content, and other indicators were measured. For the anatomical structure of the calyx tube, the detachment cells were observed. The formation time of the two groups of detached cells was compared. In the GBS simplified genome sequencing, a phylogenetic tree was constructed based on the obtained SNP sites. A principal component analysis, population genetic structure analysis, and genetic diversity index analysis were carried out. In the aspect of SSR molecular marker identification, the SSR types were counted. Polyacrylamide gel electrophoresis was performed. The results demonstrate the following: (1) the fruit-setting rate (30.87%) and calyx-removal rate (68.11%) in the YB group were significantly higher than those in the CK group (19.37%) and the calyx-removal rate (55.18%). (2) There was no significant difference in fruit quality indexes, such as average fruit weight (127.10–130.00 g) and soluble sugar content (9.47–9.56%) between the two groups. (3) Abscission-layer cells were observed at 2, 4, 6, 8, and 10 h after calyx tube discoloration in the YB group and at 48, 72, and 96 h after calyx tube discoloration in the CK group. (4) The genetic background of the YB group and the CK group was similar at the GBS level, but there were differences at the DNA level. This research finally shows that Korla fragrant pear bud mutation material is a good germplasm resource. This germplasm resource can promote the structural optimization of Korla fragrant pear varieties and the healthy development of the industry.
