Integrons are the special group of mobile genetic elements which can acquire, shuffle and spread antimicrobial resistance genes. They mediate drug resistance among Enterobacteriaceae and Non-fermenters. The present study investigation was undertaken to envisage the presence of Class I integrase gene among multidrug resistant Gram-negative bacteria. In this prospective study, 60 bacterial isolates from various clinical specimens were subjected to routine identification and susceptibility testing by conventional methods. Later the isolates were subjected for detection of intI1 gene by conventional PCR. The overall prevalence of intI1 gene among the clinical isolates was 60% (36/60) in our study. Class I integrase gene distribution among multidrug resistant bacteria was 80% (24/30) in comparison to non-multidrug resistant bacteria 43.34% (13/30). Antibiotics that were linked to Class I Integrons and shown to be statistically significant (p = 0.05) included ampicillin, aztreonam, ciprofloxacin, cefazolin, cefepime and tobramycin all showed high levels of resistance. Prevalence of intI1 gene was high among Enterobacteriaceae than Non-fermenters. There is a significant association between intI1 gene and multidrug resistance among these pathogens. Klebsiella species are highly multidrug resistant in comparison to other isolates and all of them harboured intI1 gene. Integrons can be a platform for the discovery of certain new metabolic pathways which can bring revolution in the field of antibiotic drug resistance. The information on the Integrons will aid us in prompt utilization of antimicrobial agents for the treatment.