Protein function is often regulated by posttranslational modifications (PTMs), and recent advances in mass spectrometry have resulted in an exponential increase in PTM identification. However, the functional significance of the vast majority of these modifications remains unknown. To address this problem, we compiled nearly 200,000 phosphorylation, acetylation, and ubiquitination sites from 11 eukaryotic species, including 2,500 newly identified ubiquitylation sites for Saccharomyces cerevisiae. We developed methods to prioritize the functional relevance of these PTMs by predicting those that likely participate in cross-regulatory events, regulate domain activity, or mediate protein-protein interactions. PTM conservation within domain families identifies regulatory “hot spots” that overlap with functionally important regions, a concept that we experimentally validated on the HSP70 domain family. Finally, our analysis of the evolution of PTM regulation highlights potential routes for neutral drift in regulatory interactions and suggests that only a fraction of modification sites are likely to have a significant biological role. Display omitted ► We compiled a resource of nearly 200,000 PTMs across 11 eukaryotic species ► Computational methods were developed to assign function to PTMs ► Conservation studies identify regulatory regions within domain families ► Evolutionary analysis suggests that only a fraction of PTMs are functionally important Analysis of 200,000 PTMs across 11 species in the light of structural and interaction data reveals ground rules for establishing whether a PTM is likely to impact protein function and suggests that only a fraction of modification sites are likely to be functionally important.