Abstract Background Carbapenem resistance in Gram-negative bacteria is an ongoing public health problem of global dimensions leaving very few treatment options for infected patients. Objectives To study the dissemination of plasmid-borne carbapenemase genes in Gram-negative bacteria from a diagnostic centre in Tamil Nadu, India. Methods A total of 151 non-repetitive isolates belonging to 10 genera were collected between January 2015 and December 2016 from a diagnostic centre in Tamil Nadu. The isolates included Escherichia coli (n = 57), Klebsiella pneumoniae (n = 45), Pseudomonas aeruginosa (n = 10), Salmonella Typhi (n = 8), Enterobacter cloacae (n = 8), Acinetobacter baumannii (n = 7), Serratia marcescens (n = 5), Achromobacter xylosoxidans (n = 5), Proteus mirabilis (n = 5), Klebsiella oxytoca (n = 5) and Elizabethkingia meningoseptica (n = 1). Results Of the 151 isolates, 71% (n = 107) and 68% (n = 103) were found to be resistant to meropenem and imipenem, respectively. The most prevalent β-lactamase gene was blaNDM-1 (n = 22), followed by blaOXA-181 (n = 21), blaGES-1 (n = 11), blaOXA-51 (n = 9), blaGES-9 (n = 8), blaOXA-23 (n = 7) and blaIMP-1 (n = 3). We also observed blaOXA-23 in E. coli (n = 4), and three K. pneumoniae were positive for both, blaOXA-23 and blaOXA-51. Plasmid incompatibility (inc/rep) typing results showed that the resistance genes (n = 11) were present in the isolates carrying plasmid-types IncX, IncA/C, IncFIA-FIB and IncFIIA. The plasmid-borne resistance genes in E. coli and K. pneumoniae were transferred to susceptible E. coli AB1157. Conclusions This study highlights the prevalence of carbapenem resistance and the acquisition of plasmid-borne carbapenemase genes in Gram-negative bacteria isolated at this centre.