: The green tiger prawn, Penaeus semisulcatus is one of the most important of the Penaeidae family In Persian Gulf. Based on the morphological characteristics, a new subspecies called P.semisulcatus persicus is recognized. Molecular study of the species and subspecies was considered using mitochondrial D-loop. In this study, 6 specimens (3 species and 3 subspecies ) were collected. Genomic DNA, extracted from shrimp pleopods were used as a template. Fragments of the D- loop region were amplified and sequenced using the penaeus specific primers (12S(D-loopF)/ PCR-IR(D-loopR).The sequence alignment and genetic distance were studied using Clustal W and MEGA 5.1. Results showed that the amplified D- loop fragments have 612 – 615 bp and are AT rich. The sequences alignments indicated that the D- loop sequences of Penaeus semisulcatus samples are identical (99%). Results of the present study indicated that the D - loop of P. semisulcatus persicus are the same (99%). The calculated genetic distance between species samples was 0.003 – 0.013, but, between subspecies samples was 0.002 – 0.005. The calculated genetic distance between species and samples was 0.319 – 0.325. This result suggests a significant genetic divergence between species and subspecies.