We determined almost complete nifH and nifD genes from representatives of all recognized genera of heliobacteria, the strictly anaerobic phototrophs belonging to the low GC gram-positive bacteria. The heliobacterial sequences formed a highly supported monophyletic group that is clearly distinct from any known diazotrophs, in both NifH and NifD trees. According to the classification of nitrogenase genes in four major clusters, the clade of heliobacterial sequences belonged to cluster I and did not cluster with any of the Clostridium (cluster III) or Paenibacillus (cluster I) species, the close neighbors of heliobacteria based on the 16S rRNA phylogeny. One partial anfH or alternative nitrogenase sequence was detected from Heliobacterium gestii. Although Heliophilum fasciatum is known to fix nitrogen based on the acetylene reduction test, nifH and/or nifD genes were not detected by either the PCR amplification or Southern hybridization methods.