<em>Spodoptera frugiperda</em> J.E. Smith (Lepidoptera: Noctuidae) is an invasive pest of corn plants spreading throughout the world, including East Nusa Tenggara (NTT), Indonesia. Despite the wide distribution, there is a lack of information on the strain or genetic diversity of the pest in NTT. Therefore, this study aimed to determine the strain of <em>S. frugiperda</em> from several areas in NTT with a molecular method using <em>cytochrome oxidase subunit 1</em> (<em>COI</em>)<em> </em>and <em>triose phosphate isomerase</em> (<em>Tpi</em>)<em> </em>gene markers. The<em> </em>samples were collected from 3 islands: Timor, Flores, and Sumba. Amplification of the marker genes was carried out using 3 specific primers to identify the strain obtained from samples. Subsequently, polymerase chain reaction (PCR) products were sequenced and the DNA sequences were analyzed using the BioEdit and BLAST programs. Phylogeny analyses were carried out using the MEGA 11 program to verify the strain group of samples with reference isolates from other countries found in GenBank. The PCR results showed product amplicon size 811 bp for the <em>CO1A</em> marker. Based on phylogeny tree analysis using <em>COI </em>marker, <em>S. frugiperda </em>from NTT showed 2 clades, namely corn and rice strains. The characterization results showed that <em>S. frugiperda</em> in NTT comprised 63.6% corn and 36.4% rice strains. <em>COIB </em>classified <em>S. frugiperda</em> from NTT into the h4 haplotype subgroup, while <em>Tpi </em>gene marker was in the corn strain. This study provided valuable information regarding the strain of <em>S. frugiperda</em> in Indonesia to determine the appropriate control strategy.