Physicochemical properties are potentially useful in predicting functional differences between aligned protein subfamilies. We present a method that considers physicochemical properties from ancestral sequences predicted to have given rise to the subfamilies of interest by gene duplication. Comparison between two map kinases subfamilies, p38 and ERK, revealed a region that had an excess of change in properties after gene duplication followed by conservation within the two subfamilies. This region corresponded to that experimentally defined as important for substrate and pathway specificity. The derived scores for the region of interest were found to differ significantly in their distribution compared to the rest of the protein when the Kolmogorov–Smirnov test was applied (p = 0.005). Thus, the incorporation of ancestral physicochemical properties is useful in predicting functional differences between protein subfamilies. In addition, the method was applied to the MKK and MAPK components of the p38 and JNK pathways. These proteins showed a similar pattern in their evolution and regions predicted to confer functional differences are discussed.