A C-type lectin of multiple CRDs (CfLec-4) from Chlamys farreri was selected to investigate the sequence variation and functional differentiation of its CRDs. Its four CRDs with EPD/LSD, EPN/FAD, EPN/LND and EPN/YND key motifs were recombined separately. The recombinant proteins of CRD1 and CRD2 (designated as rCRD1 and rCRD2) could bind LPS and mannan, while the recombinant proteins of CRD3 and CRD4 (designated as rCRD3 and rCRD4) could bind LPS, PGN, mannan and glucan. Moreover, rCRD3 displayed broad microbe binding spectrum towards Gram-positive bacteria Staphylococcus aureus and Micrococcus luteus, Gram-negative bacteria Escherichia coli and Vibrio anguillarum, as well as fungi Pichia pastoris and Yarrowia lipolytica. These results indicated CRD3 contributed more to CfLec-4's nonself-recognition ability. Furthermore, CRD1, CRD3 and CRD4 functioned as opsonin participating in the clearance against invaders in scallops. The sequence variation in Ca2+ binding site 2 among CRDs was suspected to be associated with such functional differentiation.