<p><i>Spodoptera frugiperda</i> (Smith), the Fall Armyworm (FAW), is a polyphagous pest that has emerged from the Western Hemisphere and feeds more than 80 distinct plant species. FAW has spread over the majority of parts in Africa and Asia in just four years, thus significantly reducing the amount of maize crop production. This nasty insect has been recently exposed in several locations around Pakistan and has threatened the production of crops. The present study focuses on diversity analysis of FAW in Pakistan based on DNA Barcoding of COI gene and expression analysis of its pesticide resistance genes. Multiple and pairwise sequence alignments of FAW COI gene sequences retrieved from NCBI revealed high similarity among the insect population of Pakistan. This finding represents the same species in the country and any future mutation, may potentially affect the entire FAW population of Pakistan. Chemical pesticides, mainly including chlorpyrifos are primarily used to control the FAW; however, non-judicial use of these chemicals would lead to the development of resistance against pesticides. Detoxification enzymes, or metabolic resistance factors, are one of the key pathways that promote resistance in the FAW. Expression analysis of CYP321B1 and CYP321A8 followed by toxicity bioassay showed that these genes are significantly overexpressed in response to chlorpyrifos exposure. Therefore, prolonged use of pesticides like Chlorpyrifos can be a significant factor of resistance development in FAW. Overall, the present study provides insights to improve management strategies for the prevention of fall armyworms in field crops.</p> Graphical Abstract <p></p>