Many cofactors and nucleotides containing sulfur atoms are known to have important functions in a variety of organisms. Recently, the biosynthetic pathways of these sulfur containing compounds have been revealed, where many enzymes relay sulfur atoms. Increasing evidence also suggests that the prokaryotic sulfur-relay enzymes might be the evolutionary origin of ubiquitination and the related systems that control a wide range of physiological processes in eukaryotic cells. However, these sulfur-relay enzymes have been studied in only a small number of organisms. Here we carried out comparative genomic analysis and examined the presence and absence of sulfurtransferases utilized in the biosynthetic pathways of molybdenum cofactor (Moco), 2-thiouridine (S(2)U), and 4-thiouridine (S(4)U), and IscS, a cysteine desulfurase. We found that all eukaryotes and many other organisms lack the intermediate enzymes in S(2)U biosynthesis. It is also found that most genes lack rhodanese homology domain (RHD), a catalytic domain of sulfurtransferase. Some organisms have a conserved sequence composed of about 100 residues in the C terminus of TusA, different from RHD. Host-associated organisms have a tendency to lose Moco biosynthetic enzymes, and some organisms have MoaD-MoaE fusion protein. Our findings suggest that sulfur-relay pathways have been so diversified that some putative sulfurtransferases possibly function in other unknown pathways.