All five species in the heterotrophic micro-algal genus Prototheca and their relatives were compared for the extent of nucleotide divergence in the nuclear small-subunit (SSU) and in the 5' end of large-subunit (LSU) ribosomal RNA genes (rDNAs). Phylogenetic analysis based on combined SSU and LSU rDNA sequence alignment was implemented with the neighbor-joining, the maximum-parsimony, and the maximum-likelihood methods. The relationships among the species of Prototheca based on this data set were largely concordant with those inferred from SSU or LSU rDNA sequences alone. The obtained phylogenetic trees indicated that P. stagnora and P. ulmea should be regarded as different species and that both of the species as well as P. moriformis were placed in a cluster represented by P. zopfii, whereas P. wickerhamii was not directly grouped together with the other members of Prototheca and was more closely related to the autotrophic alga Auxenochlorella protothecoides. Therefore, the genus Prototheca is paraphyletic in its present circumscription; and these conclusions lead us to propose the transfer of P. wickerhamii to Auxenochlorella or to a new genus. On the basis of nucleotide sequence similarities, unlike SSU rDNA, the LSU rDNA region examined in this study appeared to be variable in recognizing a heterogeneity within a single species P. zopfii, which had been shown earlier in a chemotaxonomic study.