Mobilized colistin resistance genes (mcr) may confer resistance to the last-resort antimicrobial colistin and can often be transmitted horizontally.mcrencode phosphoethanolamine transferases (PET), which are closely related to chromosomally encoded, intrinsic lipid modification PET (i-PET; e.g., EptA, EptB, CptA). To gain insight into the evolution ofmcrwithin the context of i-PET, we identified 69,814 MCR-like proteins present across 256 bacterial genera (obtained by querying known MCR family representatives against the National Center for Biotechnology Information [NCBI] non-redundant protein database via protein BLAST). We subsequently identified 125 putative novelmcr-like genes, which were located on the same contig as (i) ≥1 plasmid replicon and (ii) ≥1 additional antimicrobial resistance gene (obtained by querying the PlasmidFinder database and NCBI’s National Database of Antibiotic Resistant Organisms, respectively, via nucleotide BLAST). At 80% amino acid identity, these putative novel MCR-like proteins formed 13 clusters, five of which represented putative novel MCR families. Sequence similarity and a maximum likelihood phylogeny ofmcr, putative novelmcr-like, andipetgenes indicated that sequence similarity was insufficient to discriminatemcrfromipetgenes. A mixed-effect model of evolution (MEME) indicated that site- and branch-specific positive selection played a role in the evolution of alleles within themcr-2andmcr-9families. MEME suggested that positive selection played a role in the diversification of several residues in structurally important regions, including (i) a bridging region that connects the membrane-bound and catalytic periplasmic domains, and (ii) a periplasmic loop juxtaposing the substrate entry tunnel. Moreover,eptAandmcrwere localized within different genomic contexts. CanonicaleptAgenes were typically chromosomally encoded in an operon with a two-component regulatory system or adjacent to a TetR-type regulator. Conversely,mcrwere represented by single-gene operons or adjacent topap2anddgkA, which encode a PAP2 family lipid A phosphatase and diacylglycerol kinase, respectively. Our data suggest thateptAcan give rise to “colistin resistance genes” through various mechanisms, including mobilization, selection, and diversification of genomic context and regulatory pathways. These mechanisms likely altered gene expression levels and enzyme activity, allowingbona fide eptAto evolve to function in colistin resistance.