The extent by which different cellular components generate phenotypic diversity is an ongoing debate in evolutionary biology that is yet to be addressed by quantitative comparative studies. We conducted an in vivo mass-spectrometry study of the phosphoproteomes of three yeast species (Saccharomyces cerevisiae, Candida albicans, and Schizosaccharomyces pombe) in order to quantify the evolutionary rate of change of phosphorylation. We estimate that kinase-substrate interactions change, at most, two orders of magnitude more slowly than transcription factor (TF)-promoter interactions. Our computational analysis linking kinases to putative substrates recapitulates known phosphoregulation events and provides putative evolutionary histories for the kinase regulation of protein complexes across 11 yeast species. To validate these trends, we used the E-MAP approach to analyze over 2,000 quantitative genetic interactions in S. cerevisiae and Sc. pombe, which demonstrated that protein kinases, and to a greater extent TFs, show lower than average conservation of genetic interactions. We propose therefore that protein kinases are an important source of phenotypic diversity. Natural selection at a population level requires phenotypic diversity, which at the molecular level arises by mutation of the genome of each individual. What kinds of changes at the level of the DNA are most important for the generation of phenotypic differences remains a fundamental question in evolutionary biology. One well-studied source of phenotypic diversity is mutation in gene regulatory regions that results in changes in gene expression, but what proportion of phenotypic diversity is due to such mutations is not entirely clear. We investigated the relative contribution to phenotypic diversity of mutations in protein-coding regions compared to mutations in gene regulatory sequences. Given the important regulatory role played by phosphorylation across biological systems, we focused on mutations in protein-coding regions that alter protein-protein interactions involved in the binding of kinases to their substrate proteins. We studied the evolution of this "phosphoregulation" by analyzing the in vivo complement of phosphorylated proteins (the "phosphoproteome") in three highly diverged yeast species—the budding yeast Saccharomyces cerevisiae, the pathogenic yeast Candida albicans, and the fission yeast Schizosaccharomyces pombe—and integrating those data with existing data on thousands of known genetic interactions from S. cerevisiae and Sc. pombe. We show that kinase-substrate interactions are altered at a rate that is at most two orders of magnitude slower than the alteration of transcription factor (TF)-promoter interactions, whereas TFs and kinases both show a faster than average rate of functional divergence estimated by the cross-species analysis of genetic interactions. Our data provide a quantitative estimate of the relative frequencies of different kinds of functionally relevant mutations and demonstrate that, like mutations in gene regulatory regions, mutations that result in changes in kinase-substrate interactions are an important source of phenotypic diversity.