Based on two mitochondrial DNA sequences; cytochrome b and 16S rRNA with using a total DNA template from muscle tissues of two puffer fishes species Arothron hispidus and Tetraodon lineatus, our study was performed to reveal           a general view about the phylogenetic linkages among River Nile Tetraodon lineatus and Red Sea Arothron hispidus to other marine puffer fishes. The nucleated sequence lengths based on cytochrome b gene in Arothron hispidus and Tetraodon lineatus were (337 and 367 bp. respectively), while using the 16S gene in Arothron hispidus and Tetraodon lineatus revealed nucleated sequence of length (186 and 237 bp. respectively). The sequenced regions of cytochrome b in Arothron hispidus and Tetraodon lineatus were submitted in the GenBank/NCBI under accession numbers MN186251 and MN186252 respectively, also the sequenced regions of 16S in Arothron hispidus and Tetraodon lineatus were submitted in the GenBank/NCBI under accession numbers MN186287 and MN175976 respectively. The phylogenetic linkages were designed using three phylogenetic methods; Maximum Likelihood (ML), Neighbour Joining (NJ) and Minimum Evolution (ME). The Pairwise genetic distances of both (cyt-b) and (16S) illustrated that, the Nile puffer fish Tetraodon lineatus was closely related to marine puffer fishesof genera Arothron and Canthigaster more than other marine puffer fish genera. Our results showed high efficiency of cyt-b and 16S rRNA in the phylogenetic analysis, so we strongly support the usefulness of them for taxonomy studies of puffer fishes.