Background: This present study objectives are to identify and determine the prevalence of ESBLs producing gram-negative isolates from cases of neonatal sepsis that occurred in Indore, Madhya Pradesh; and also to investigate the genotypic characterization of ESBL producing gram negative bacilli isolated in blood culture of neonatal sepsis. Methods: The research is experimental and descriptive. Both are investigated. It was carried out after the Index Medical College Hospital & Research Centre (IMCH&RC) Institutional Ethical Committee approved it. Eligibility: The Index Medical College Hospital & Research Centre in Indore performed blood cultures on newborns with sepsis. Drawing neonatal blood. Cultured blood was tested. The study included inborn and out born infants admitted to the IMCH and RC's Neonatal Intensive Care Unit (NICU) with suspected clinical features of sepsis at admission or after admission for other reasons. NICU and IMCH/RC infants were studied. Despite uncertainty, the infants received antibiotics. Results: The detection of each blaOXA, blaCTX-M, and blaTEM ESBL genes was significantly associated with an ESBL phenotypes. The strength of association varied by the specific ESBL genes, with the odds ratio (OR) of 1.4 (95% CI: 1.2-1.6, p<0.05) for blaOXA, 3.6 (95% CI: 2.8-3.8, p<0.001) for blaTEM, and 14.7 (95% CI: 11.4-18.8, p<0.001) for blaCTXM in the Fisher’s test. Conclusion: The findings of this study indicated that there was a high likelihood for GNB to be MDR and ESBL positive whenever any of the blaTEM, blaCTXM, or blaOXA genes were identified. In addition, the identification of the blaCTXM genes provided strong evidence that the GNB were resistant to the betalactam antimicrobials with an extended spectrum of activity