Pseudouridine (Ψ) modifications are the most abundant RNA modifications; however, their distribution and functional significance in bacteria remain largely unexplored compared to eukaryotic systems. In this study, we present the first transcriptome-wide and quantitative mapping of Ψ modifications across five diverse bacterial species (Bacillus cereus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Pseudomonas syringae) at single-base resolution, utilizing the optimized BID-