King fish (Scomberomorus commerson) belonging to the family scombridae considered ecologically and economically important fish species. This research was carried out to identify molecular identification and genetic assessment using mtDNA D-Loop via HRM-Real Time PCR. A total of 100 Dorsal fin clip samples of Scomberomorus commerson were sampled from four locations along Persian Gulf (Bushehr & Doha), Oman Sea (Jask) and Arabian Sea (Karachi) and after isolating genomic DNA, HRM-Real Time PCR was conducted with the use of SYTO 9 saturating dye to classify samples. Representative of groups to the waters of Bushehr, Doha, Jask and Karachi were 8, 6, 9 and 6, respectively. Ultimately, 20 groups were identified by using High Resolution Melting Analysis. Representatives of groups were sequenced after conducting anew PCR. The results of sequencing were inferred by performing blast on NCBI and Scomberomorus commerson were identified. Thereafter, distinctions were observed constructing phylogenetic tree via ensued sequences. Likewise, considerations demonstrated that High Resolution melting-Real Time PCR not just could be an effectual technique to accelerate enquiry procedure but, isolate and classify manifold samples for populations enquiry.