The EF-hand is a helix-loop-helix motif observed mainly in intracellular calcium binding proteins. The EF-hand usually occurs as a pair, EF-lobe, which is a unit of evolution and structure. Penta EF-hand (PEF) proteins form a unique group including calpain, sorcin, grancalcin, ALG-2, and peflin. The fifth EF-hand of PEF proteins makes a pair with that of another PEF protein. The members of PEF family have diverse functions and their evolution is complex. The interaction of PEF proteins with target occurs at several sites. Here, we analyzed the ancestral sequences of each group of PEF proteins and determined the interfaces for the specific and selective interaction to the target among several PEF proteins. The shape of the groove for interaction at common site is different among PEF proteins. We found that the changes at limited sites induced the divergence of interaction sites that determines the selectivity of targets. The residues involved the changes at limited sites are important for the drug design selective to each PEF protein.