Prunus × yedoensis ‘Someiyoshino’ is the most popular cultivar of flowering cherry in Japan. Although the origin of this cultivar has been considered hybrid between P. pendula f. ascendens and P. lannesiana var. speciosa, the paternity of P. lannesiana has not been clearly proven by molecular analysis. To reveal the origin of ‘Somei-yoshino,’ we analyzed sequences of intron 19 and exon 20 of PolA1, a single-copy nuclear gene encoding the largest subunit of RNA polymerase I. One of two exon 20 sequences found in ‘Somei-yoshino’ was the same as that of P. pendula, whereas the other sequence was shared with several taxa in seven wild species, including P. jamasakura and P. lannesiana. ‘Somei- yoshino’ contained two different haplotypes of the intron 19 sequences; one was the same as that of P. lannesiana, which is endemic to the Izu and Boso Peninsula in Japan. While another haplotype of ‘Somei-yoshino’ was different from that of P. pendula by two SNPs but identical to one of two haplotypes of P. pendula ‘Komatsuotome,’ which is a cultivar found in the Ueno Park, Tokyo. These results indicated that ‘Someiyoshino’ probably originated by the hybridization of cultivars derived from P. pendula and P. lannesiana.