Mitochondrial markers such as , COI, 16S rRNA and D loop have been used for population genetics and phylogenetic relationships between shrimps. Recently, mitochondrial 16S rRNA/tRNA val region was found to contain more variable sites than the 16S rRNA and COI region. Based on morphological characteristics, a new subspecies called P.semisulcatus persicus is recognized. Therefore, molecular study of the species and subspecies was considered using mitochondrial 16SrRNA/tRNA val region. In this study,6 specimens (3 species and 3 subspecies ) were collected. Genomic DNA extracted from shrimp pleopods were used as template. Fragments ofthe 16SrRNA/tRNA val region were amplified and sequenced using 16ScruF/16ScruR primers. The sequence alignment and genetic distance were studied using ClustalW and MEGA 5.1. Results showed that the amplified 16SrRNA/tRNA val region have 522 -525 bp and are AT rich. The sequences alignments indicated, that with the exception of Per4, species and subspecies samples were identical(99%). Calculated geneticdistance between Per4 and other samples was 0.074 – 0.079. This results revealed that like COI, there is no significance difference between samples of penaeus semisulcatus and subspecies in the 16SrRNA/tRNA val region. Significant genetic differences between Per4 and other samples, possibly due to non-harmful mutations accumulate over successive generations in the this area of mitochondria.