In this study, ancient genome analysis of two Yayoi human skeletons excavated from the Shimomotoyama Rock Shelter in Sasebo city was carried out. To judge by the morphological study, these individuals were descendants of the indigenous Jomon people rather than of an immigrant Yayoi people. However, as a result of DNA analysis using the next generation sequencer, it was revealed that these individuals have the genome of both Jomon and immigrant Yayoi people. These human bones belong to the end of the Yayoi period. From the results of this study, it was revealed that at this time, in the surrounding areas of Northwest Kyushu Island, the admixture was quite advanced between the indigenous Jomon and immigrant Yayoi people. These results indicate that it is necessary to reconsider the relationship between these two groups who have been thought to have been completely different. The present study also showed that the data obtained by analyzing the nuclear genome of ancient human bones is effective for capturing the situation of such a mixed population. Further analyses of more archaeological sites dating from the Yayoi period of Kyushu Island will lead us toward a more precise genetic characterization of these societies.