AbstractThe functional evolution of proteins advances through gene duplication followed by functional drift, whereas molecular evolution occurs through random mutational events. Over time, protein active‐site structures or functional epitopes remain highly conserved, which enables relationships to be inferred between distant orthologs or paralogs. In this study, we present the first functional clustering and evolutionary analysis of the RCSB Protein Data Bank (RCSB PDB) based on similarities between active‐site structures. All of the ligand‐bound proteins within the RCSB PDB were scored using our Comparison of Protein Active‐site Structures (CPASS) software and database (http://cpass.unl.edu/). Principal component analysis was then used to identify 4431 representative structures to construct a phylogenetic tree based on the CPASS comparative scores (http://itol.embl.de/shared/jcatazaro). The resulting phylogenetic tree identified a sequential, step‐wise evolution of protein active‐sites and provides novel insights into the emergence of protein function or changes in substrate specificity based on subtle changes in geometry and amino acid composition.