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Antibiotic use has risen sharply in recent decades, fueling resistance. Since China reported colistin resistance in 2015, global concern has grown. While few cases of colistin resistance have been reported in Enterobacteriaceae from Pakistan, molecular factors remain unclear. This study aimed to determine the prevalence of colistin-resistant Enterobacteriaceae (ColRE) in a tertiary care hospital in Abbottabad, Pakistan. Altogether 89 Enterobacteriaceae isolates were collected from in-patients or out-patients at Ayub Teaching Hospital Abbottabad. The isolates were identified using API 20E kit and antibiotic susceptibility testing was performed following CLSI guidelines. Molecular screening of antibiotic resistance genes was conducted by PCR. The results showed E. coli (40.4%) as the most common pathogen, followed by K. pneumoniae (21.3%), S. typhi (19%), Enterobacter (9%), Raoultella ornithinolytica (4.4%), K. aerogenes (2.2%), Citrobacter (2.2%) and K. oxytoca (1%). Antibiotic testing showed highest resistance to aztreonam and cefepime (65.1%), and lowest to colistin (29.2%) and meropenem (22.4%). Overall, 48.3% of the isolates were multidrug resistant (MDR), with K. pneumoniae showing the highest MDR rate (68.4%). Molecular screening of colistin resistance genes found 15.3% of isolates positive for mcr-1 or mcr-2 genes. Simultaneous presence of mcr-1 and mcr-2 was identified in 3.8% of isolates. The study also found the first case of K. aerogenes with mcr-1 positivity. In conclusion, the study reveals a high rate of colistin resistance Enterobacteriaceae with notable presence of plasmid mediated genes. The study recommends nationwide surveillance of ColRE, with emphasis on the screening of plasmid-mediated determinants.