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Not AvailableIn this study, a multiplex PCR assay was developed for the authentication of three commercially important shrimp species viz., Penaeus monodon, Penaeus vannamei, and Penaeus indicus. Three new species-specific primers designed from the mitochondrial 16S rRNA gene yielded distinct amplicon size of 360, 277, and 203 bp for P. monodon, P.vannamei, and P.indicus, respectively. The sensitivity, accuracy, specificity, and suitability of the multiplex PCR assay was determined as per standard protocol. The assay exhibited high specificity to the target shrimp species. The sensitivity ranged from 1 to 0.1 ng/μl of DNA concentration. In-house validation with eleven types of mixed-species gave 100% correlation in the authentication of the target shrimp species. Field validation with 22 processed commercial shrimp products revealed 22% mislabeling. The developed multiplex PCR assay authenticated the three commercially important Penaeids in processed shrimp products from other related shrimp, fish as well as crab species in a single run.Not Availabl