The three previously recognized genera of ‘ Nitrosolobus’, Nitrosospira and ‘ Nitrosovibrio’ were combined into one genus, Nitrosospira, on the basis of 16S rDNA sequence similarities. However, this classification has been controversial for some time, since the marked differences in their shapes suggest that they are not closely related. In this study, the phylogenetic analyses of the three groups using two genotypical markers, glyceraldehyde-3-phosphate dehydrogenase (GAP, gap), and 3-phosphoglycerate kinase (PGK, pgk), were performed. In the phylogenetic tree inferred from gap and pgk, the three genera appeared as clearly separated clusters. This is the first study of markers that are able to reveal the precise phylogenetic relationship among ‘ Nitrosolobus’, Nitrosospira and ‘ Nitrosovibrio’.