This study aimed to reveal the evolutionary timescale and processes underlying the diversity of Rubus in the Japanese Archipelago. We conducted molecular phylogenetic analyses of most native species (35 species), along with previously published data from 116 foreign species, based on nuclear ribosomal internal transcribed spacer (ITS) and chloroplast DNA sequences. Most of the northern species of Japan, that is, R. chamaemorus, R. pedatus, R. vernus, R. pseudojaponicus, and R. ikenoensis, were found to belong to anciently diverged lineages; in particular, R. ikenoensis formed a unique lineage distinct from other species. The other species diverged into two evolutionary groups. One included subg. Malachobatus, Chamaebatus, and sects. Pungentes, Idaeanthi, and Parvifolii (subg. Idaeobatus), which was further divided into two clades in the chloroplast phylogenies. Although the phylogenetic structures within this group were unresolved, R. sieboldii has been proven to be recently derived. The second group represented a well-supported clade, comprising sects. Microphylli, Corchorifolii, Peltati, and Rosifolii (subg. Idaeobatus), and suggested early Miocene diversification of this Asian lineage associated with character specialization in vegetative reproduction and leaf shape. This clade was further resolved into lower clades primarily representing the sectional classifications, although the placement of the earliest diverged species, R. sumatranus, R. peltatus, R. corchorifolius, and R. chingii, was incongruent among gene trees. At the lower taxonomic levels, R. illecebrosus, R. grayanus, and the thornless species of sect. Microphylli showed earlier divergence.