In Japan, there are many genetic resources for breeding hydrangea cultivars, but it is difficult to utilize them effectively for breeding because of a lack of phylogenetic information. In this study, the phylogenetic relationship of H. macrophylla (Thunb.) Ser. f. normalis (E.H.Wilson) H.Hara and H. serrata (Thunb.) Ser. was evaluated by using RAPD markers and sequences of the plastid genes rbcL and matK. The materials were collected from their wild populations throughout Japan. Both RAPD analysis and chloroplast DNA analysis indicated that the genetic diversity of H. serrata var. serrata was higher than that of H. macrophylla f. normalis or that of H. serrata (Thunb.) Ser. var. yesoensis (Koidz.) H.Ohba. These analyses revealed that H. serrata var. serrata of Japan was separated into two groups; i.e., eastern serrata group and western serrata group. The western serrata group was divided into two or three subgroups by single base substitutions in the matK or rbcL fragment sequences. The results of chloroplast DNA analysis indicated that H. serrata of Shikoku, which was one of the western serrata subgroups, was evolutionarily differentiated from other western serrata subgroups. MatK and rbcL sequences of the eastern serrata group were identical to those of H. macrophylla f. normalis and H. serrata var. yesoensis. The matK sequences of the eastern serrata group, H. macrophylla f. normalis and H. serrata var. yesoensis, contained a duplication of 6 bp (GGTTAT), which was not found in the western serrata group or other Hydrangea species. Analysis of the matK and rbcL sequences revealed that H. serrata var. serrata is paraphyletic and that the eastern serrata group, H. macrophylla f. normalis and H. serrata var. yesoensis, form a monophyletic group. The present study provided useful information for breeding hydrangea cultivars and for the taxonomic treatment of H. macrophylla and H. serrata including the varieties.