The fall armyworm (FAW), an invasive pest of maize, is an emerging threat in Southern Asia after America and Europe. Recently, this notorious pest has also been found in different areas of Pakistan. To assess its presence in Pakistan, a survey was carried out in the provinces of Punjab, Sindh, and Khyber Pakhtunkhwa during May–October 2019. We observed the highest incidence of FAW in Sindh with maximum impact in districts Tando-Allahyar and Hyderabad. These samples were identified as Spodoptera frugiperda on the morphological and taxonomical bases. However, morphological identification of this pest is very difficult at early larval instars. Here, we use the mitochondrial cytochrome c oxidase I (COI) gene region for the precise identification of larva of this invasive pest at species level. Two different regions of COI gene (COI-5′ and COI-3′) were used as molecular markers for the identification of this species. DNA sequence similarity searches of the obtained COI gene sequences (NCBI GenBank Accession Nos. MW241537, MW241538, MW349515, MW349516, MW349493 and MW349494) revealed that genetically it is more than 99% identical to S. frugiperda. The phylogenetic analysis indicated it as the rice-strain (R-strain). Both 3′- and 5′-fragment tree topologies showed that the collected samples of the FAW species belong to the R-strain. To the best of our knowledge, this is the first report providing molecular evidence for the existence of R-strain of S. frugiperda that was found feeding on maize crop in Sindh, Pakistan, using COI gene sequences as a marker.