Abstract In Japan, honey bees, bumble bees, and flies are used in strawberry production to produce normally shaped fruits. These insects may determine the inbreeding or outbreeding of strawberry flowers. Whole-genome DNA of the strawberry cultivar ‘Shizutakara’ showed no amplification of DNA fragments when PCR was performed with a F3H CAPS DNA marker. However, in other ordinary strawberry cultivars, amplification of DNA fragments was observed on PCR with the F3H. Thus, growing one plant of ‘Shizutakara’ with other cultivars and a single insect species, harvesting ‘Shizutakara’ fruit, and performing DNA analysis of the seeds could show whether the seed resulted from inbreeding or outbreeding, as the DNA fragments would be amplified from outbred but not from inbred seed. On this basis, differences in pollination mode among these three pollinators were analyzed. DNA analysis showed that DNA fragments could not be detected in seeds obtained by fly pollination in the greenhouse. Flies appeared to promote inbreeding. For honey bees and bumble bee, amplified DNA fragments could be detected. Some seeds pollinated by honey bees and bumble bee appeared to be of outbred origin. From these results, differences in pollination mode among insect pollinators were identified.