Bacterial flagellin protein is a potent microbe-associated molecular pattern. Immune responses are triggered by a 22-amino-acid epitope derived from flagellin, known as flg22, upon detection by the pattern recognition receptor FLAGELLIN-SENSING2 (FLS2) in multiple plant species. However, increasing evidence suggests that flg22 epitopes of several bacterial species are not universally immunogenic to plants. We investigated whether flg22 immunogenicity systematically differs between classes of the phylum , using a dataset of 2,470 flg22 sequences. To predict which species encode highly immunogenic flg22 epitopes, we queried a custom motif ( STxxDNDNxAGxxI ) in the flg22 sequences, followed by sequence conservation analysis and protein structural modeling. These data led us to hypothesize that most flg22 epitopes of the γ- and β- are highly immunogenic, whereas most flg22 epitopes of the α-, δ-, and ε- are weakly to moderately immunogenic. To test this hypothesis, we generated synthetic peptides representative of the flg22 epitopes of each proteobacterial class, and we monitored their ability to elicit an immune response in . The flg22 peptides of γ- and β- triggered strong oxidative bursts, whereas peptides from the ε-, δ-, and α- triggered moderate, weak, or no response, respectively. These data suggest flg22 immunogenicity is not highly conserved across the phylum . We postulate that sequence divergence of each taxonomic class was present prior to the evolution of FLS2, and that the ligand specificity of FLS2 was driven by the flg22 epitopes of the γ- and β- , a monophyletic group containing many common phytopathogens.Formula: see text Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.