The rapid increase in number and diversity of Extended Spectrum β-Lactamases (ESBLs) producing Enterobacteriaceae in natural aquatic environment is a major health concern worldwide. This study investigates abundance and distribution of ESBL producing multidrug resistant Enterobacteriaceae and molecular characterization of ESBL genes among isolates from highly polluted stretch of river Yamuna, India. Water samples were collected from ten different sites distributed across Delhi stretch of river Yamuna, during 2014-15. A total of 506 non duplicate Enterobacteriaceae isolates were obtained. Phenotypic detection of ESBL production and antibiotic sensitivity for 15 different antibiotics were performed according to CLSI guidelines (Clinical and Laboratory Standard Institute, 2015). A subset of ESBL positive Enterobacteriaceae isolates were identified by 16S rRNA gene and screened for ESBL genes, such as bla_CTX-M, bla_TEM and bla_OXA. Out of 506 non-duplicate bacterial isolates obtained, 175 (34.58%) were positive for ESBL production. Susceptibility pattern for fifteen antibiotics used in this study revealed higher resistance to cefazolin, rifampicin and ampicillin. A high proportion (76.57%) of ESBL positive isolates showed multidrug resistance phenotype, with MAR index of 0.39 at Buddha Vihar and Old Delhi Railway bridge sampling site. Identification and PCR based characterization of ESBL genes revealed the prevalence of bla_CTX-M and bla_TEM genes to be 88.33% and 61.66%, respectively. Co-occurrence of bla_CTX-M and bla_TEM genes was detected in 58.33% of the resistant bacteria. The bla_OXA gene was not detected in any isolates. This study highlights deteriorating condition of urban aquatic environment due to rising level of ESBL producing Enterobacteriaceae with multidrug resistance phenotype.