Protein phosphorylation is a crucial post-translational modification in bacteria, but has not been extensively studied because of the technical difficulty of phosphopeptide enrichment. We present a new enrichment protocol, approximately 10 times more efficient than conventional approaches in E. coli. This protocol also performed well in B. subtilis and K. pneumoniae, in terms of high coverage and phosphopeptide identification numbers. Moreover, three high-confidence Ser/Thr phosphorylation motifs as well as 29 other motifs at various confidence levels were discovered for the first time, implying that both the position of phospho-acceptor residues and the surrounding sequences are critical for kinase-substrate specificity. As for N-terminal phosphorylation, a low rate of co-occurrence of N-terminal acetylation and acidic residues at the antepenultimate position appears to be prokaryote-specific. The comprehensive prokaryotic phosphoproteomes generated by our new protocol suggest the existence of distinct phosphorylation preferences between prokaryotes and eukaryotes.