20-Hydroxyecdyone (20E), an active form of ecdysteroid, is the key hormone in insect growth and development. To date five P450 enzymes involved in the last four steps of ecdysteroid biosynthesis from ketodiol to 20E namely Spook (Spo), Phantom (Phm), Disembodied (Dib), Shadow (Sad) and Shade (Shd) related to ecdysteroid biosynthesis, are identified and the character of last four enzymes is well studied in Drosophila melanogaster, Bombyx mori and Manduca sexta. All of these genes are called Halloween genes. In this study, we extended these works to a major pest insect in agriculture in India, the cotton leafworm Spodoptera litura (Lepidoptera: Noctuidae). We identified the sequence of five Halloween genes partially and converted into amino acid sequences and then three of them were compared with a very near African species of Spodoptera, Spodoptera littoralis. The results suggest amino acid substitution in open reading frame of phantom, shade and disembodied protein in Spodoptera litura.