Abstractl-Asparaginases, which convert l-asparagine to l-aspartate and ammonia, come in five types, AI-AV. Some bacterial type AII enzymes are a key element in the treatment of acute lymphoblastic leukemia in children, but new l-asparaginases with better therapeutic properties are urgently needed. Here, we search publicly available bacterial genomes to annotate l-asparaginase proteins belonging to the five known types. We characterize taxonomic, phylogenetic, and genomic patterns of l-asparaginase occurrences pointing to frequent horizontal gene transfer (HGT) events, also occurring multiple times in the same recipient species. We show that the reference AV gene, encoding a protein originally found and structurally studied in Rhizobium etli, was acquired via HGT from Burkholderia. We also describe the sequence variability of the five l-asparaginase types and map the conservation levels on the experimental or predicted structures of the reference enzymes, finding the most conserved residues in the protein core near the active site, and the most variable ones on the protein surface. Additionally, we highlight the most common sequence features of bacterial AII proteins that may aid in selecting therapeutic l-asparaginases. Finally, we point to taxonomic units of bacteria that do not contain recognizable sequences of any of the known l-asparaginase types, implying that those microorganisms most likely contain new, as yet unknown types of l-asparaginases. Such novel enzymes, when properly identified and characterized, could hold promise as antileukemic drugs.