Carboxylesterase-mediated insecticide resistance was studied in Spodoptera litura Fab with three different populations from Varanasi, Delhi and Sonepat. The level of susceptibility to pyrethroid insecticides (cypermethrin and bifenthrin), activity of carboxylesterase (CarE) and amino acid substitutions in esterase gene of each population were investigated. Insecticide bioassay revealed that Sonepat and Delhi populations of S. litura were 2.2- and 2.1-fold resistant to cypermethrin and bifenthrin, respectively. Delhi and Sonepat populations had elevated activity of enzyme level of 26.47±0.21 and 28.02±0.05, respectively. The Varanasi population (10.09 ± 0.10 μmol/min/mg of protein) exhibited lowest CarE activity. The CarE gene of 500 bp fragment that encodes 167 amino acid residues was characterised in all populations. Cluster analysis of gene sequences revealed that all three populations were 92 to 95% identity and were closely related to other Spodoptera spp. Among the 167 amino acid residues, 27 were identical in all Spodoptera species and seven substitutions were observed. Sequence alignment confirmed no substitution at conserved position and one semiconserved substitution. In the characterised region, the substitution W224S which is responsible for CarE activity reduction in insecticide resistant Culex pipiens was not observed in any of the Spodoptera populations. A strong positive relation (r = 0.99) was observed between the CarE activity and determined LC50. Thus, elevated CarE activity in Delhi and Sonepat field populations was attributed to pyrethroid resistance. Metabolic detoxification mainly appears to be due to overproduction of CarE enzyme in respective populations and not because of amino acid residue changes.