We analyzed sequence variation in chloroplast DNA (cpDNA) to investigate the origin of the cultivated strawberry, Fragaria ×ananassa. From analysis of two noncoding regions, trnL–trnF and trnR–rrn5, we found three haplotypes (V, C, and X) in F. ×ananassa. Haplotype V corresponded to the haplotype of F. virginiana and was possessed by cultivars bred over a wide geographic range, including North America, Europe, and Japan. Almost all the North American cultivars analyzed in this study possessed haplotype V, suggesting a founder effect. Haplotype C corresponded to the haplotype of F. chiloensis and was detected mainly in Japanese cultivars. Haplotype X was found in only two English cultivars. This haplotype was positioned as intermediate between haplotypes V and C in a median-joining network and was considered to be representative of the process of differentiation between F. virginiana and F. chiloensis. Results of controlled crosses indicate that cpDNA haplotypes of F. ×ananassa are maternally inherited. These results verify that F. ×ananassa is an interspecific hybrid between F. virginiana and F. chiloensis and indicate that traditional cultivars of F. ×ananassa have been derived from at least three maternal lineages. We demonstrate that the cpDNA variation detected in this study can be used to verify parentage and for extending hypotheses about June yellows, a leaf variegation disorder in strawberry.