A diverse family of PDZ domains has been identified, but the rules that govern their ligand specificity are not clear. Here we propose a novel classification of PDZ domains based on the nature of amino acids in the two critical positions in the PDZ domain fold. Using these principles, we classified PDZ domains present in the SMART database. Using yeast two-hybrid, in vitro pull-down and plasmon surface resonance assays, we demonstrated that in agreement with their position in the proposed classification the Mint1-1, hINADL-5, and PAR6 PDZ domains display similar dual ligand specificity. The proposed classification helps to organize PDZ domain containing proteins.