Vitamin B??, a cobalt-containing micronutrient, has been shown to limit phytoplankton growth in the Ross Sea of the Southern Ocean. However, B?? biosynthesis potential in this environment remains uncharacterized. Select bacteria and archaea synthesize B?? while many phytoplankton require it for growth. Low ratios of bacterial biomass production to primary productivity and high concentrations of labile cobalt in Antarctic surface water suggest that factors controlling bacterial growth rather than cobalt availability may determine vitamin production rates here. In order to assess B?? biosynthesis potential, degenerate polymerase chain reaction primers were designed to target the genetic locus cbiA/cobB, encoding cobyrinic acid a,c-diamide synthase, a B?? biosynthesis protein. Sequencing the DNA compliment of Ross Sea 16S rRNA (see Supporting information) allowed targeting of cbiA/cobB probes to dominant bacterial groups. CbiA/cobB DNA sequences were successfully identified in clone libraries from the Ross Sea. To our knowledge, this study represents the first targeted molecular characterization of environmental B?? biosynthesis potential. A newly identified group of cbiA/cobB sequences dominated the diversity of the sequences retrieved; their expression was confirmed via mass spectrometry-based peptide detection. These sequences seem to have originated from a previously undescribed group of bacteria that could dominate the B?? biosynthesizing community in polar systems.