We analyzed the phylogenetic relationships of 49 specimens comprising 14 morphologically similar species of Pucciniastrum distributed in Japan based on the sequence data of the large subunit rDNA (D1/D2), 5.8S rDNA, and internal transcribed spacer (ITS) regions. Neighbor-joining and parsimony analyses generated six major groups for both the D1/D2 and ITS regions. Pucciniastrum circaeae and P. epilobii formed a single group. P. hydrangeae-petiolaris, P. coryli, P. fagi, P. hikosanense, P. tiliae, and P. boehmeriae were each a distinct clade, and P. fagi formed a close relationship with P. hikosanense. However, these analyses did not support the monophyly of the following species: P. kusanoi, P. actinidiae, P. corni, P. styracinum, P. yoshinagai, and P. miyabeanum.