Background The subfamily Plusiinae is an economically important moth pest group under species rich family Noctuidae (Lepidoptera). The evolutionary history of this important subfamily has not been resolved completely. Particularly in India, the genus is represented by a species complex, but the taxonomic delineation between them is still unclear.Methods and results In present study, a comprehensive phylogenetic relationship among Indian species of this subfamily has been inferred for the first time based on mitochondrial marker, Cytochrome Oxidase I (COI), and nuclear gene marker, Ribosomal Protein S5 (RPS5), emphasizing tribal level classification. Here, we analyzed 125 plusiinae taxa from eight biogeographical zones of India comprising 2 molecular markers: mitochondrial marker, Cytochrome Oxidase I (COI), and nuclear gene marker, Ribosomal Protein S5 (RPS5). The results revealed that Plusiinae tribes were monophyletic and considered sister groups that shared many derived characteristics. The ML/MP cladogram based on the barcoding gene successfully separates all the species, but not all tribes. While the nuclear gene marker, RPS5 separated all the species according to their tribes. The combined analysis of both genes showed tribe resolution into distinct clades.Conclusions This is the first comprehensive study on phylogenetic studies of 25 species of plusiinae from India which gives a clear information about species position and arrangement within taxa.