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DNA barcoding is one of the powerful DNA-based identification tools that are used for accurate identification of a species. Despite several DNA barcodes were used, the accuracy and suitability of these barcodes depend on speciesspecific variations.The current study was conducted to compare the efficiency of two mitochondrial genes to the nuclear Internal Transcribed Spacer (ITS) region in the molecular identification of genus Dicentrarchus. A total of 80 fish samples for Dicentrarchus labrax and Dicentrarchus punctatus were randomly collected from two different locations in Egypt; Alexandria and Bardawil Lagoon. All samples were morphologically characterized. For species barcoding, the ITS region was firstly employed to carry out the PCR amplifications. Additionally, two mitochondrial genes; cytochrome b (Cyt b) and cytochrome oxidase subunit I (COI) were also used for species barcoding. To evaluate the efficiency of each marker, three different approaches were used. Firstly, phylogenetic relationship was constructed between the collected samples and a reference species using each genetic marker. Secondly, Automated Barcode Gap Discovery (ABGD) method was used for each marker in order to assign the samples into presumed species without priori species assumption. Lastly, the Poisson Tree Processes (PTP) model was used which relies on Bayesian support values to delimit species on the input tree. The two species of genus Dicentrarchus exhibited nearly similar ITS sequences, leading to an ambiguous identification of the two species. However, the two mitochondrial COI and Cyt b genes were able to accurately distinguish between the two species. The three approaches, phylogeny, ABGD and PTP presented consistent results. Overall, COI and Cyt b outperformed ITS in assigning species accurately. Mitochondrial barcodes could provide a leading guide for fish species identification. ITS should be abandoned in favor of COI and Cyt b as primary DNA barcode markers for fish species in general and Dicentrarchus genus, in particular.