Mitochondrial genome differentiation in the genus Phyllostachys was investigated by restriction fragment length polymorphism (RFLP) analyses of the restriction fragment patterns or Southern hybridization patterns of endonuclease-treated mitochondrial (mt) DNA. First, intraspecific variation of mtDNA in 3 species, P. pubescens, P. nigra and P. bambusoides, was studied using a large number of samples collected from different locations in Japan. Little intraspecific differentiation was detected in P. nigra and P. bambusoides, whereas P. pubescens showed some intraspecific variation. Second, the restriction fragment patterns and Southern hybridization patterns of mtDNAs of 13 Phyllostachys species were analyzed. Their comparison indicated that P. nigra and P. dulcis, and P. angusta and P. propinqua have the same mitochondrial genome, respectively. The restriction patterns allowed the identification of 9 out of 13 species. Based on the percentage of common restriction fragments, all the species except for P. aureosulcata were clustered into the following 3 groups by the UPGMA method; (1) P. angusta, P. propinqua, P. pubescens and P. praecox, (2) P. nigra, P. dulcis, P. humilis and P. aurea, and (3) P. bambusoides, P. bissetii, P. viridis and P. makinoi. Clustering of 13 species based on the results of Southern pattern analysis led to the identification of the following 4 groups; (1) P. angusta, P. propinqua, P. aureosulcata, P. pubescens, P. praecox and P. bambusoides, (2) P. nigra and P. dulcis, (3) P. bissetii, P. viridis and P. makinoi, and (4) P. humilis and P. aurea. The results of the 2 methods differed in the following aspects; (1) affiliation of P. bambusoides differed, (2) cluster 2 in the restriction pattern analysis was divided into 2 clusters by Southern pattern analysis, and (3) intercluster relationships were considerably different between the 2 dendrograms. To develop reliable phylogenetic relationships in the genus Phyllostachys, it is necessary to increase the number of restriction enzymes used in the restriction pattern analysis as well as the number of probes used in the Southern pattern analysis.