In order to clarify the current phylogeny of the haloarchaea, particularly the closely related genera that have been difficult to sort out using 16S rRNA gene sequences, the DNA-dependent RNA polymerase subunit B′ gene (rpoB′) was used as a complementary molecular marker. Partial sequences of the gene were determined from 16 strains of the family Halobacteriaceae. Comparisons of phylogenetic trees inferred from the gene and protein sequences as well as from corresponding 16S rRNA gene sequences suggested that species of the genera Natrialba, Natronococcus, Halobiforma, Natronobacterium, Natronorubrum, Natrinema/Haloterrigena and Natronolimnobius formed a monophyletic group in all trees. In the RpoB′ protein tree, the alkaliphilic species Natrialba chahannaoensis, Natrialba hulunbeirensis and Natrialba magadii formed a tight group, while the neutrophilic species Natrialba asiatica formed a separate group with species of the genera Natronorubrum and Natronolimnobius. Species of the genus Natronorubrum were split into two groups in both the rpoB′ gene and protein trees. The most important advantage of the use of the rpoB′ gene over the 16S rRNA gene is that sequences of the former are highly conserved amongst species of the family Halobacteriaceae. All sequences determined so far can be aligned unambiguously without any gaps. On the other hand, gaps are necessary at 49 positions in the inner part of the alignment of 16S rRNA gene sequences. The rpoB′ gene and protein sequences can be used as an excellent alternative molecular marker in phylogenetic analysis of the Halobacteriaceae.