Spodoptera spp. (Lepidoptera: Noctuidae) are highly polyphagous pests that inflict serious damage to a wide spectrum of crops. The ability of Spodoptera spp. to thrive on diverse host plants is an adaptive advantage for their survival in the ecosystem, which is achieved by its high mobility, fecundity and capacity to develop resistance to wide spectrum of chemical insecticides. In this study, we present molecular diversity and phylogenetic relationship of S. litura (Fabricius) and S. exigua (Hübner) inferred from mitochondrial cytochrome oxidase-I (COI). Alignment of the sequences of COI from various life stages of the 2 species of Spodoptera shows that the molecular identification is independent of life stages and polymorphism of the target species. Maximum likelihood analyses of S. litura, S. exigua and S. mauritia (Boisduval) reveal that there exist significant variations among these. Spodoptera exigua showed intraspecific variations with respect to different geographic locations. Present study proves the utility of COI for identification of S. litura and S. exigua irrespective of their life stages, and also draws inferences on the phylogenetic relationships between the 3 pest species.