ABSTRACT Polymyxins are last-resort antimicrobial agents used to treat infections caused by carbapenem-resistant Enterobacteriaceae . Due to the worldwide dissemination of polymyxin resistance in animal and human isolates, we aimed to characterize polymyxin resistance associated with the presence of mcr-1 in Enterobacteriaceae and nonfermenter Gram-negative bacilli, using isolates collected retrospectively in Colombia from 2002 to 2016. A total of 5,887 Gram-negative clinical isolates were studied, and 513 were found to be resistant to the polymyxins. Susceptibility to colistin was confirmed by broth microdilution for all mcr-1 -positive isolates, and these were further subjected to whole-genome sequencing (WGS). The localization of mcr-1 was confirmed by S1 pulsed-field gel electrophoresis (S1-PFGE) and CeuI-PFGE hybridization. Transferability was evaluated by mating assays. A total of 12 colistin-resistant isolates recovered after 2013 harbored mcr-1 , including 8 Escherichia coli , 3 Salmonella enterica serovar Typhimurium, and 1 Klebsiella pneumoniae isolate . E. coli isolates were unrelated by PFGE and belonged to 7 different sequence types (STs) and phylogroups. S . Typhimurium and K. pneumoniae isolates belonged to ST34 and ST307, respectively. The mcr-1 gene was plasmid borne in all isolates but two E. coli isolates which harbored it on the chromosome. Conjugation of mcr-1 was successful in 8 of 10 isolates (8.2 × 10 −5 to 2.07 × 10 −1 cell per recipient). Plasmid sequences showed that the mcr-1 plasmids belonged to four different Inc groups (a new IncP-1 variant and the IncFII, IncHI1, and IncH families). Our results indicate that mcr-1 is circulating in clinical isolates of colistin-resistant Enterobacteriaceae in Colombia and is mainly harbored in transferable plasmids.