This study describes a polymerase chain reaction using restriction fragment length polymorphism (PCR-RFLP) assay based on the 16S rRNA mitochondrial gene to identify commercial food products of wide range of Penaeidae and Pandalidae shrimp species commercialised in the Tunisian market. Phylogenetic analyses on 16S rRNA mitochondrial gene were used to study the relationships among the considered species. Penaeidae shrimp species was easily differentiated and confirmed by direct sequencing, showing a genetic distance of 0.34 with respect to Pandalidae species. A rapid and reliable PCR method using restriction fragment length polymorphism (RFLP) with three restriction enzymes (HpyCH4III/ MboI / AluI) was optimized for unambiguous differentiation of shrimp from 19 commercial market samples (raw and processed products). Results showed that the restriction fragment length polymorphism technique can be used to identify Tunisian shrimp species and thus to control not only commercial fraud but also efficient restocking program.