We have developed a semiempirical algorithm called Family Values (FamVal), which identifies residues that encode functional specificity in a protein sequence. Given a multiple sequence alignment (MSA) grouped into functionally distinct subfamilies, FamVal calculates a specificity score for each subfamily at every amino acid position of an MSA. This algorithm was used to predict specificity-encoding positions within the tetramerization assembly (T1) domain of voltage-gated potassium (Kv) channel subfamilies Kv3 and Kv4. The importance of one such position (Arg to Ala at MSA position 93) was confirmed by in vitro pull-down assays. The structural basis of this assembly discrimination was elucidated by determining the crystal structure of the Kv4 T1 domain and comparing it to the Kv3 T1 domain.