Many regulatory proteins bind peptide regions of target proteins and modulate their activity. Such regulatory proteins can often interact with highly diverse target peptides. In many instances， it is not known if the peptide-binding interface discriminates targets in a biological context， or whether biological specificity is achieved exclusively through external factors such as subcellular localization. We used an evolutionary biochemical approach to distinguish these possibilities for two such low-specificity proteins: S100A5 and S100A6. We used isothermal titration calorimetry to study the binding of peptides with diverse sequence and biochemistry to human S100A5 and S100A6. These proteins bound distinct， but overlapping， sets of peptide targets. We then studied the peptide binding properties of orthologs sampled from across five amniote species. Binding specificity was conserved along all lineages， for the last 320 million years， despite the low specificity of each protein. We used ancestral sequence reconstruction to determine the binding specificity of the last common ancestor of the paralogs. The ancestor bound the entire set of peptides bound by modern S100A5 and S100A6 proteins， suggesting that paralog specificity evolved via subfunctionalization. To rule out the possibility that specificity is conserved because it is difficult to modify， we identified a single historical mutation that， when reverted in human S100A5， gave it the ability to bind an S100A6-specific peptide. These results reveal strong evolutionary constraints on peptide binding specificity. Despite being able to bind a large number of targets， the specificity of S100 peptide interfaces is likely important for the biology of these proteins.