DNA Barcoding is a new taxonomic approach for identifying biological specimens and managing species diversity. Intraspecific variation in a sequence is in order of magnitude less than that observed inter-specifically and this provides the means by which species are differentiated. It is also being used as a research tool for refining the understanding of biological diversity, and as a system for assigning biological samples to their species of origin. The objective of this study was to investigate the applicability of DNA barcoding as a tool for species identification of the green tiger prawn, Penaeus semisulcatus. The present study on molecular identification based on DNA barcoding of MT-COI gene was also aimed to construct a possible molecular phylogenetic tree with selected shrimp species for understanding their evolutionary relationship/ significance. Penaeid shrimps are an important resource in crustacean fisheries, representing more than the half of the gross production of shrimp worldwide. The results showed that DNA barcodes present a high degree of interspecies variation making classification possible even at the species level and this technique is relatively cheap, fast, and useful in the identification of incorrectly labelled market products.