O-methyltransferase (OMT), a protein present ubiquitously in wide range of organisms plays significant role in methylation of small macro molecules for various functional and regulatory purposes. In crustaceans, OMT has functional role in growth, reproduction, ovarian development and molting. In the present study, suppression subtractive hybridization (SSH) performed using gill tissues of low (3ppt) and high (55ppt) salinity stressed shrimp Penaeus monodon resulted in identification of differentially expressed genes involved in signal transduction pathways, metabolism, defense proteins, DNA repair and synthesis, apoptosis, cell cycle regulation along with unknown and hypothetical proteins. Catechol-O-methyltransferase (COMT) a type of OMT was identified by SSH as one of the differentially expressed genes of shrimp P. monodon subjected to low and high salinity stress. The full length cDNA of COMT was cloned from the gills of P. monodon which consisted an open reading frame of 666 bp, encoding 221 amino acids. The ORF revealed one each of N-glycosylation and O-glycosylation sites and nine phosphorylation sites. The deduced amino acid sequence of COMT exhibited high sequence identity (92%) with COMT class of protein from Fenneropenaeus chinensis. Real time PCR analysis of the shrimp samples exposed to low salinity conditions at 3ppt revealed significant increase in expression of COMT transcripts in the guts at 24 h, 48 h, 1 week and 2 weeks, gills at 24 h and in the muscle tissues at 48 h, with maximum expression of the COMT levels by 5 fold in guts (1 week), 1 fold in gills (24 h) and 1.5 fold in muscle (48 h) respectively. The increased expression level of COMT at different time intervals in different tissues suggests a possible role of this gene in salinity stress tolerance in shrimps under low salinity conditions.