Dissimilatory nitrate reduction to ammonium (DNRA) and denitrification are contrasting microbial processes in the terrestrial nitrogen (N) cycle, in that the former promotes N retention and the latter leads to N loss (i.e., the formation of gaseous products). The nitrite reductase NrfA catalyzes nitrite reduction to ammonium, the enzyme associated with respiratory nitrite ammonification and the key step in DNRA. Although well studied biochemically, the diversity and phylogeny of this enzyme had not been rigorously analyzed. A phylogenetic analysis of 272 full-length NrfA protein sequences distinguished 18 NrfA clades with robust statistical support (>90% Bayesian posterior probabilities). Three clades possessed a CXXCH motif in the first heme-binding domain, whereas all other clades had a CXXCK motif in this location. The analysis further identified a KXRH or KXQH motif between the third and fourth heme-binding motifs as a conserved and diagnostic feature of all pentaheme NrfA proteins. PCR primers targeting a portion of the heme-binding motifs that flank this diagnostic region yielded the expected 250-bp-long amplicons with template DNA from eight pure cultures and 16 new nrfA-containing isolates. nrfA amplicons obtained with template DNA from two geomorphically distinct agricultural soils could be assigned to one of the 18 NrfA clades, providing support for this expanded classification. The extended NrfA phylogeny revealed novel diagnostic features of DNRA populations and will be useful to assess nitrate/nitrite fate in natural and engineered ecosystems.