In order to understand the evolutionary aspects of the chloroplast DNA (cpDNA) structures in Rosaceous plants, a physical map of peach (Prunus persica cv. Hakuhou) cpDNA was constructed. Fourteen lambda phage clones which covered the entire sequence of the peach cpDNA were digested by restriction enzymes (SalI, XhoI, BamHI, SacI, and PstI) used singly or in combination. The molecular size of peach cpDNA was estimated to be about 152 kb. The gene order and contents were revealed to be equivalent to those of standard type of angiosperms by the localization of 31 genes on the physical map. Eighteen accessions from 14 Prunus species (P. persica, P. mira, P. davidiana, P. cerasis, P. cerasifera, P. domestica, P. insititia, P. spinosa, P. salicina, P. maritima, P. armeniaca, P. mume, P. tomentosa, P. zippeliana, and P. salicifolia) and one interspecific hybrid were used for the structural analysis of cpDNAs. Seventeen mutations (16 recognition site changes and one length mutation) were found in the cpDNA of these 18 accessions by RFLP analysis allowing a classification into 11 genome types. Although the base substitution rate in the recognition site (100p = 0.72) of cpDNA in Prunus was similar to that of other plants, i.e., Triticum-Aegilops, Brassica, and Pisum, it differed from Pyrus (100p = 0.15) in Rosaceae. Seven mutations including one length mutation were densely located within a region of about 9.1 kb which includes psbA and atpA in the left border of a large single-copy region of Prunus cpDNAs. The length mutation was detected only in P. persica and consisted of a 277 bp deletion which occurred in a spacer region between the trnS and trnG genes within the 9.1 kb region. Additional fragment length mutations (insertion/deletion), which were not detected by RFLP analysis, were revealed by PCR and sequence analyses in P. zippeliana and P. salicifolia. All of these length mutations occurred within the 9.1 kb region between psbA and atpA. This region could be an intra-molecular recombinational hotspot in Prunus species.