Background: Colistin is one of the oldest antibiotics in the polymyxin group, and is used mostly against gramnegative bacteria. Because of developing resistance among clinical isolates colistin has become an alternative drug for multidrug resistant bacteria. Objectives: To determine colistin resistance among isolates from Tamil Nadu, India. Methods: We included 94 gram-negative isolates from two centers in Tamil Nadu in the present study. Isolates were identified by 16S rRNA sequencing. Minimal inhibitory concentrations (MICs) were determined by agar dilution. Results: The isolates identified at species level included 48 Escherichia coli, 9 Klebsiella pneumoniae, 10 Pseudomonas aeruginosa, 5 Proteus mirabilis, 4 Salmonella enterica, 3 Enterobacter hormaechei, 3 Enterobacter cloacae, 2 Achromobacter xylosoxidans, 2 Acinetobacter baumannii, 1 Providencia vermicola, 1 Acinetobacter towneri, 1 Enterobacter gergoviae, 2 Providencia rettgeri, 1 Enterobacter asburiae, 1 Pseudomonas stutzeri, and 1 Salmonella typhi. The MIC of colistin ranged from 0.12 μg/ml to 128 μg/ml. The MIC50was 1 μg/mL and MIC90was >128 μg/ml. The MIC > 8 μg/mL was resistant breakpoint for all the species. A total of 27 isolates were resistant to colistin. Colistin resistant isolates included E. coli (9/48), K. pneumoniae (6/9), P. aeruginosa (3/10), A. baumannii (1/2), P. mirabilis (4/5), E. cloacae (1/3), P. rettgeri (2/2), and S. enterica (1/4). Carbapenem susceptibility of colistin resistant isolates was tested and 14 were found to be resistant to meropenem. Conclusions: Our study indicates the emergence of colistin resistant isolates from clinical samples among different groups of gram-negative organisms. Resistance to both carbapenem and colistin occurs. Developing new antibiotics and programs to reduce nosocomial infections is necessary especially for multidrug resistant isolates.