Type II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins, crucial for the catabolic metabolism, because they contribute to the maintenance of the NADH/NAD+ balance. In several pathogenic bacteria and protists, NDH-2s are the only enzymes performing respiratory NADH:quinone oxidoreductase activity. For this reason and for being considered absent in mammals, NDH-2s were proposed as suitable targets for novel antimicrobial therapies. We selected all sequences of genes encoding NDH-2s from fully sequenced genomes present in the KEGG database. These genes were present in 61% of the 1805 species belonging to Eukarya (83%), Bacteria (60%) and Archaea (32%). Notably sequences from mammal species including humans were retrieved in our selection as NDH-2s. The data obtained and the already available information allowed systematizing several properties of NDH-2s: (i) the existence of additional sequence motifs with putative regulatory functions, (ii) specificity towards NADH or NADPH and (iii) the type of quinone binding motif. We observed that NDH-2 family distribution is not congruent with the taxonomic tree, suggesting different origins for the eukaryotic sequences and possible lateral gene transfer among prokaryotes. We note the absence of genes coding for NDH-2 in anaerobic phyla and the presence of multiple copies in several genomes, specifically in cyanobacteria. These observations inspired us to propose a metabolic hypothesis for the appearance of NDH-2s.