[Objective] The biosynthesis of ε-poly-L-lysine is controlled by the ε-poly-L-lysine synthetase. This paper aims to study the distribution and sequence features of Pls. [Methods] Pls proteins were predicted in the completely sequenced genomes via identification of the substrate-recognition and condensation domains and amino acid residues that determine the substrate specificity. [Results] One hundred and thirteen Pls were identified from 110 genomes, mostly distributed in Actinobacteria, with two identified in Gram-negative bacteria. Most Pls from closely related species display a high degree of identity. [Conclusion] Pls may be widely distributed in Actinobacteria. The adenylation, thiolation, and condensation domains of Pls are conserved while the transmembrane domains and linkers show otherwise.