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Objectives: Historically, colistin has been considered a last-line therapeutic option against multidrug-resistant Gram-negative bacterial infections. However, chromosomally-encoded and plasmid-mediated colistin resistance is increasingly being reported worldwide. Spread of the plasmid-borne colistin resistance gene mcr-1 is of great concern since it can be transferred between bacteria. The aim of this study was to investigate the prevalence of mcr-1 in Escherichia coli and Klebsiella pneumoniae collected from human clinical specimens in Thailand during 2014-2017., Methods: Minimum inhibitory concentrations (MICs) of colistin were determined by the broth microdilution method for 317 non-duplicate Enterobacteriaceae clinical isolates (37 E. coli and 280 K. pneumoniae). All isolates were screened for the mcr-1 gene by PCR., Results: The colistin MIC 50 , MIC 90 and MIC range for the 37 E. coli isolates were 0.5, 8 and 0.5-32mg/L, respectively. The mcr-1 gene was detected in 11 E. coli isolates (29.7%). Escherichia coli harbouring the mcr-1 gene had a colistin MIC range of 4-32mg/L. The colistin MIC 50 , MIC 90 , and MIC range for the 280 K. pneumoniae isolates were 32, >128, and 0.25 to >128mg/L, respectively. The mcr-1 gene was detected in 4 K. pneumoniae isolates (1.4%). Klebsiella pneumoniae harbouring the mcr-1 gene had a colistin MIC range of 4-64mg/L., Conclusions: This is the first report on the prevalence of the mcr-1 gene in colistin-resistant E. coli and K. pneumoniae isolated from humans in Thailand. These data provide added insight into the mechanism of colistin resistance among Enterobacteriaceae pathogens., (Copyright © 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)