New Delhi Metallo-β-lactamase-1(NDM-1) is an enzyme that hydrolyzes a wide range of β-lactams antibiotics, including carbapenems. The presence of the NDM-1 inhibits the potential of β-lactam antibiotics in treating infections caused by bacterial strains carrying such resistances, thus leaving minimal treatment options available. Due to this, the rapid distribution of NDM-1 harboring bacteria accounts for a significant public health menace worldwide. These bacteria have been detected in clinical specimens and environmental compartments where bacterial infections are ubiquitous. In this study, identification and absolute quantification of NDM-1 in sixteen lake sediment samples collected in and around Hyderabad, India, was carried out using a real-time quantitative polymerase chain reaction (qPCR), and results were expressed in gene copy number/ng (nanogram) of template DNA. Thirteen samples (out of sixteen) displayed a positive signal for NDM-1 during the qPCR analysis with the highest gene copy number/ng of template DNA (71.8) being observed in the Amberpet STP. Three samples, samples those from Durgamcheru lake, Kandi lake, and Singur dam, were negative for the NDM-1 during the qPCR analysis. Hierarchical clustering analysis was performed to categorize the sampling location into different clusters based on pollution sources and the observed results were expressed in the form of a dendrogram. © 2022 University of Tehran. All rights reserved.