To investigate how different species or ploidy level of pollen donors affects the fruit quality of kiwifruit, flowers of ‘Hayward’ kiwifruit (a hexaploidActinidia deliciosacultivar, 6x) were hand-pollinated with pollen from ten different male donors. Kiwifruit plants pollinated with four distant species—M7 (2x,A. kolomikta), M8 (4x,A. arguta), M9 (4x,A. melanandra), and M10 (2x,A. eriantha)—had a low fruit-setting rate and therefore were not investigated further. Of the other six treatments, kiwifruit plants pollinated with M4 (4x,A. chinensis), M5 (6x,A. deliciosa) M6 (6x,A. deliciosa) had a larger fruit size and weight than those pollinated with M1 (2x,A. chinensis) and M2 (2x,A. chinensis). However, pollination with M1 (2x) and M2 (2x) resulted in seedless fruits, having few small and aborted seeds. Notably, these seedless fruits had higher fructose, glucose, and total sugar and lower citric acid content. This resulted in a higher sugar to acid ratio compared to fruits from plants pollinated with M3 (4x,A. chinensis), M4 (4x), M5 (6x), and M6 (6x). Most volatile compounds increased in the M1 (2x)- and M2 (2x)-pollinated fruit. A combination of principal component analysis (PCA), electronic tongue, and electronic nose suggested that the different pollen donors significantly affected the kiwifruit’s overall taste and volatiles. Specifically, two diploid donors had the most positive contribution. This was in agreement with the findings from the sensory evaluation. In conclusion, the present study showed that the pollen donor affected the seed development, taste, and flavor quality of ‘Hayward’ kiwifruit. This provides useful information for improving the fruit quality and breeding of seedless kiwifruit.