Fruit seedlessness is a major excellent agronomic trait of citrus, in order to clarify the cause of seedlessness of ‘Dongshi Zaoyou’ pummelo. Taking ‘Dongshi Zaoyou’ pummelo and its hybrid offspring as the main test materials, we explored the causes of seedlessness of ‘Dongshi Zaoyou’ pummelo by using the staining methods of magenta acetate and aniline blue, cross-pollination and molecular markers. The results showed that ‘Dongshi Zaoyou’ pummelo was observed to have complete floral structures, pollen viability and pollen germination rates of 94.80% and 86.62%, respectively. ‘Dongshi Zaoyou’ pummelo showed self-incompatibility, whereas crosses with ‘Shatianyou’ pummelo showed cross-compatibility. A total of 164 F1 hybrids were identified from 175 cross progenies of ‘Shatianyou’ pummelo × ‘Dongshi Zaoyou’ pummelo using S5/S6-genotyped molecular markers, which excluded the possibility of male sterility in ‘Dongshi Zaoyou’ pummelo. In addition, the fruit set percentage of ‘Dongshi Zaoyou’ pummelo was 13.04% and 25.56% for two consecutive years with stimulating parthenocarpy and natural parthenocarpy, respectively. It was inferred that the self-incompatibility of ‘Dongshi Zaoyou’ pummelo and its ability to produce a partial parthenocarpy were the main reasons for its seedlessness.