© The Author(s), 2014. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Genome Biology and Evolution 7 (2015): 457-464, doi:10.1093/gbe/evu289. ; The uptake and transport of vitamin B12 (cobalamin; Cbl) in mammals involves a refined system with three evolutionarily related transporters: transcobalamin 1 (Tcn1), transcobalamin 2 (Tcn2), and the gastric intrinsic factor (Gif). Teleosts have a single documented binder with intermediate features to the human counterparts. Consequently, it has been proposed that the expansion of Cbl binders occurred after the separation of Actinopterygians. Here, we demonstrate that the diversification of this gene family took place earlier in gnathostome ancestry. Our data indicates the presence of single copy orthologs of the Sarcopterygii/Tetrapoda duplicates Tcn1 and Gif, and Tcn2, in Chondrichthyes. In addition, a highly divergent Cbl binder was found in the Elasmobranchii. We unveil a complex scenario forged by genome, tandem duplications and lineage-specific gene loss. Our findings suggest that from an ancestral transporter, exhibiting large spectrum and high affinity binding, highly specific Cbl transporters emerged through gene duplication and mutations at the binding pocket. ; This work was funded by the Fundação para a Ciência e a Tecnologia (FCT) project PEst-C/MAR/LA0015/2013. PhD grant SFRH/BD/84238/2012 awarded to M.L-.M. Postdoctoral grant SFRH/BPD/72519/2010 awarded to R.R.