The blood cells of ascidians accumulate extremely high levels of the transition metal vanadium. We previously isolated four vanadium-binding proteins (Vanabins 1–4) and a homologous protein (VanabinP) from the vanadium-rich ascidian Ascidia sydneiensis samea. In the present study, we identified cDNAs encoding five different Vanabin2-related proteins in A. sydneiensis samea blood cells. It was notable that the sequences of the encoded proteins vary from that of Vanabin2 at up to 14 specific positions, while both the polypeptide length and the 18 cysteine residues were completely conserved. The most divergent protein, named 14MT, differed from Vanabin2 at all 14 positions. Using immobilized metal-ion affinity chromatography, we found that Vanabin2 and 14MT have the same metal ion selectivity, but the overall affinity of 14MT for VO2+ is higher than that of Vanabin2. Binding number for VO2+ ions was same between Vanabin2 and 14MT as assessed by gel filtration. These results suggested that sequence variations were under strict evolutionary constraints and high affinity binding sites for VO2+ are conserved among Vanabin2 variants.