Despite the increasing reliance on polymyxin antibiotics (polymyxin B and colistin) for treatment of multidrug-resistant Gram-negative infections, many clinical laboratories are unable to perform susceptibility testing due to the lack of accurate and reliable methods. Although gradient agar diffusion is commonly performed for other antimicrobials, its use for polymyxins is discouraged due to poor performance characteristics. Performing gradient agar diffusion with calcium enhancement of susceptibility testing media has been shown to improve the identification of polymyxin-resistant isolates with plasmid-mediated resistance (mcr-1). We therefore sought to evaluate the broad clinical applicability of this approach for colistin susceptibility testing by assessing a large and diverse collection of resistant and susceptible patient isolates collected from multiple U.S. medical centers. Among 217 isolates, the overall categorical and essential agreement for calcium-enhanced gradient agar diffusion were 73.7% and 65.5%, respectively, compared to the results for reference broth microdilution. Performance varied significantly by organism group, with agreement being highest for Enterobacterales and lowest for Pseudomonas aeruginosa. Nevertheless, even for Enterobacterales, there was a high rate of very major errors (9.2%). Performance was similarly poor for calcium-enhanced broth microdilution. While calcium enhancement did allow for more accurate categorization of mcr-1-resistant isolates, there were unacceptably high rates of errors for both susceptible and non-mcr-1-resistant isolates, raising serious doubts about the suitability of these calcium-enhanced methods for routine colistin susceptibility testing in clinical laboratories.