Protein–protein binding events mediate many critical biological functions in the cell. Typically, functionally important sites in proteins can be well identified by considering sequence conservation. However, protein–protein interaction sites exhibit higher sequence variation than other functional regions, such as catalytic sites of enzymes. Consequently, the mutational behavior leading to weak sequence conservation poses significant challenges to the protein–protein interaction site prediction. Here, we present a phylogenetic framework to capture critical sequence variations that favor the selection of residues essential for protein–protein binding. Through the comprehensive analysis of diverse protein families, we show that protein binding interfaces exhibit distinct amino acid substitution as compared with other surface residues. On the basis of this analysis, we have developed a novel method, BindML, which utilizes the substitution models to predict protein–protein binding sites of protein with unknown interacting partners. BindML estimates the likelihood that a phylogenetic tree of a local surface region in a query protein structure follows the substitution patterns of protein binding interface and nonbinding surfaces. BindML is shown to perform well compared to alternative methods for protein binding interface prediction. The methodology developed in this study is very versatile in the sense that it can be generally applied for predicting other types of functional sites, such as DNA, RNA, and membrane binding sites in proteins. Proteins 2012. © 2011 Wiley Periodicals, Inc.