Extended-spectrum β-lactamases (ESBLs) are rapidly evolving group of β-lactamase enzymes produced by the Gram negative bacteria. In this study, we determined the antimicrobial sensitivity pattern of Escherichia coli isolates and prevalence of TEM, SHV and CTX-M genes in ESBL positive E. coli isolated from the patients admitted to a tertiary care hospital in North-East India. A total of 85 multidrug-resistant isolates of E. coli obtained from clinical samples; urine (n = 80), sputum (n = 3), body fluid (n = 1), vaginal discharge (n = 1) were screened for resistance to third generation cephalosporins. ESBL production in resistant isolates was determined by double disk synergy test (DDST) and phenotypic confirmatory test (PCT). ESBL positive isolates were subjected to PCR for detection of TEM, SHV and CTX-M genes. Imipenem was found to be most effective against E. coli (susceptible isolates 96.47%) while ciprofloxacin was the least effective antibiotic (resistant isolates 60%). Among 33 ESBL positive isolates confirmed via PCT, preponderance in female population (60.6%) was noted. The most prevalent gene was bla(SHV) (63.04%) followed by bla(TEM) and bla(CTX-M) (60.86 and 54.34%, respectively) in ESBL positive E. coli. Most of the extensively used antibiotics, appear to be ineffective against the ever-mutating bacteria. This resistance urges cautious antimicrobial management on priority. Further, it helps in effectively designing the chemotherapeutic regimen for patients of a particular geographic area.