This study aimed to the detection of extended spectrum beta-lactamases by phenotypic and genotypic methods in nosocomial Gram-negative isolates. A number of 194 samples from hospitalized patients (urine, burn swab, surgical swab, and sputum) were collected These patients were at Al-Hussein and Al-Nasiriyah teaching hospitals in Al-Nasiriyah city. ESBLs enzymes were detected phenotypically by ceftazidime (30 ?g) and cefotaxime (30 ?g) (Screening test) and Double Disk Synergy Test (DDST) (Confirming test). The results showed that out of 109 isolates 89 (81.65%) were Gram negative bacilli. A total of 68 Gram-negative were ?-lactamases producer. Moreover, the results of screening test recorded that 55 (80.88%) of isolates were resistant to cefotaxime and ceftazidime, while 17 (25%) isolates produced ESBLs by (DDST).