Lepidoptera is the second most diverse insect order outnumbered only by the Coeleptera. Acetylcholinesterase (AChE) is the major target site for insecticides. Extensive use of insecticides, to inhibit the function of this enzyme, have resulted in the development of insecticide resistance. Complete knowledge of the target proteins is very important to know the cause of resistance. Computational annotation of insect acetylcholinesterase can be helpful for the characterization of this important protein. Acetylcholinesterase of fourteen lepidopteran insect pest species was annotated by using different bioinformatics tools. AChE in all the species was hydrophilic and thermostable. All the species showed lower values for instability index except L. orbonalis, S. exigua and T. absoluta . Highest percentage of Arg, Asp, Asn, Gln and Cys were recorded in P. rapae . High percentage of Cys and Gln might be reason for insecticide resistance development in P. rapae. Phylogenetic analysis revealed the AChE in T. absoluta , L. orbonalis and S. exigua are closely related and emerged from same primary branch. Three functional motifs were predicted in eleven species while only two were found in L. orbonalis, S. exigua and T. absoluta. AChE in eleven species followed secretory pathway and have signal peptides. No signal peptides were predicted for S. exigua , L. orbonalis and T. absoluta and follow non secretory pathway. Arginine methylation and cysteine palmotylation was found in all species except S. exigua , L. orbonalis and T. absoluta . Glycosylphosphatidylinositol (GPI) anchor was predicted in only nine species.