New Delhi metallo-β-lactamase-1 (NDM-1), mostly carried by Gram negative Enterobacteriaceae, is one of the leading causes of healthcare associated infections worldwide. It is capable of inactivating all β-lactam antibiotics except aztreonam and is encoded by the blaNDM-1 gene. The gene is reported to be located on bacterial chromosomes and plasmids, and transferred with the involvement of transposable elements. The current study focuses on the prevalence and association of insertion sequence (IS) with blaNDM-1 gene amongst various species of bacteria. In silico analysis showed high correlation between blaNDM-1 gene and three ISs (ISAba125, ISEc33 & ISSen4). Hence, analysis of the presence of these ISs was attempted using clinical isolates obtained from a tertiary care hospital setting in South India. Among 120 clinical isolates analyzed (PCR based detection), 51 carried the blaNDM-1 gene, out of which 39 isolates possessed at least one of the three ISs mentioned, with ISAba125 (53%) being the most common. The presence of all three ISs was detected in three NDM positive isolates. The blaNDM-1 gene could be a potential threat because of its ability to resist wide range of antibiotics and requires bold and decisive global action to resist its transferability.