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Background: Antibiotics are widely used in animals and human for treatment without referring an antibiogram that resulted into the proliferation of ESBL producing strains. Nowadays resistance to β-lactam groups of antibiotic is expanding rapidly worldwide and threatening the public healthcare due to limited treatment options. Therefore this study aimed to detect ESBL resistance genes in E. coli and Klebsiella spp. isolated from various sources of cattle in this area of study. Methods: Total 240 samples were collected during August, 2019 to June, 2020, from two districts of Eastern plain zone of Uttar Pradesh. E. coli and Klebsiella spp. isolates were confirmed using uidA and 16S rRNA gene respectively. In vitro antibiotic sensitivity test was performed using disc diffusion method. ESBL producing isolates was confirmed by DDST, ESBL E-strip and PCR analysis by targeting (bla-CTX-M-1, bla-CTX-M-9, bla-TEM and bla-SHV) genes. Result: PCR analysis of these isolates confirmed 135(56.25%) as E. coli and 16(6.67%) as Klebsiella spp. Antibiotics found to be most resistant were ampicillin (89.40%) followed by cefotaxime (72.18%), cefpodoxime (71.52%), ceftriaxone (66.9%), aztreonam (61.0%) and ceftazidime (54.30%). Total 101(66.88%) isolates were confirmed as ESBL producers using DDST and 92(60.92%) by ESBL E-strip test. ESBL genes were detected in 87(57.61%) isolates by PCR analysis and among them, bla-CTX-M-1 was found most dominant gene. [ABSTRACT FROM AUTHOR]