Abstract Background: Polyglutamine regions (polyQ) are one of the most studied and prevalent homorepeats in eukaryotes. They have a particular length-dependent codon usage, which relates to a characteristic CAG-slippage mechanism. Pathologically expanded tracts of polyQ are known to form aggregates and are involved in the development of several human neurodegenerative diseases. The non-pathogenic function of polyQ is to mediate protein-protein interactions via a coiled-coil pairing with an interactor. They are usually located in a helical context. Results: Here we study the stability of polyQ regions in evolution, using a set of 60 proteomes from four distinct taxonomic groups (Insecta, Teleostei, Sauria and Mammalia). The polyQ regions can be distinctly grouped in three categories based on their evolutionary stability: stable, unstable by length variation (inserted), and unstable by mutations (mutated). PolyQ regions in these categories can be significantly distinguished by their glutamine codon usage, and we show that the CAG-slippage mechanism is predominant in inserted polyQ of Sauria and Mammalia. The polyQ amino acid context is also influenced by the polyQ stability, with a higher proportion of proline residues around inserted polyQ. By studying the secondary structure of the sequences surrounding polyQ regions, we found that regarding the structural conformation around a polyQ, its stability category is more relevant than its taxonomic information. The protein-protein interaction capacity of a polyQ is also affected by its stability, as stable polyQ have more interactors than unstable polyQ.Conclusions: Our results show that apart from the sequence of a polyQ, information about its orthologous sequences is needed to assess its function. Codon usage, amino acid context, structural conformation and the protein-protein interaction capacity of polyQ from all studied taxa critically depend on the region stability. There are however some taxa-specific polyQ features that override this importance. We conclude that a taxa-driven evolutionary analysis is of the highest importance for the comprehensive study of any feature of polyglutamine regions.