The Sultanate of Oman has a coastline of 3,165 kilometers, comprising the Arabian Gulf, the Sea of Oman, and the Arabian Sea, making it a center for marine biodiversity in the Arabian Peninsula. The Sparidae family (Sea bream) is of commercial importance for fisheries, as they contribute significantly to local consumption and exports. However, accurate morphological identification remains challenging for some economically important species. Molecular-based identification, or DNA barcoding, has recently proven useful for identifying many fish species. Moreover, the wide distribution of some species across a wide range of the Omani coastline warrants exploring the molecular diversity associated with the adaptive potential to face current and future environmental changes. Eighteen Sparidae species have been reported in Omani waters. Here, we used DNA barcoding with cytochrome oxidase subunit І (COI, 506 bp), and 16S ribosomal RNA (16S rRNA, 521 bp), to identify 63 samples collected from the Arabian Gulf (Khasab), the Sea of Oman (Suhar), and the Arabian Sea (Salalah), representing 12 Sparidae. 16S rRNA enabled the identification of sixteen species successfully, while COI identified only ten species. The highest number of species were from the Arabian Sea (Salalah, 15), followed by the Sea of Oman (Suhar, 5), and the least from the Arabian Gulf (Khasab, 2). Two species, namely Sparus aurata and Dentex gibbosus, were recorded for the first time in Omani waters. Phylogenetic analysis revealed two genetically distinct groups. Genetic distances between species ranged from 0.011 to 0.279 based on 16S rRNA and from 0.104 to 0.259 based on COI. The results provided insight into Sparidae species' biodiversity and a representative reference library for DNA barcoding in Oman and the surrounding region. Potential population differentiation is anticipated in Sparidae species distributed across the Arabian Gulf, the Sea of Oman, and the Arabian Sea, due to geographical, environmental, and biological factors. Among the Sparidae species, A. spinifer (Forsskål, 1775) (i.e., King soldier bream) is widely distributed, commercially-important, and relatively well investigated in the Arabian Gulf and the Sea of Oman. We evaluated the relationship between the weight, length, and relative condition factor of five A. spinifer populations. The results revealed a negative allometric coefficient (b 3) across all populations, with populations from the Arabian Sea (Masirah and Salalah) showing the highest values, while populations from the Arabian Gulf (Khasab) and the Sea of Oman (Suhar) showed the lowest values. The relative condition factor (Kr) was greater than one (1) in the populations from the Arabian Sea (Salalah, Masirah), and the Arabian Gulf (Khasab), indicating better relative body condition. However, the relative condition factor in the Sea of Oman (Suhar and Muscat) indicated a lower-than-average mass. The detected differences implicated potential genetic and/or environmental factors. The genetic diversity and connectivity between A. spinifer populations along the coast of Oman were investigated using ten polymorphic microsatellite DNA markers. A total of 300 samples from five locations were analyzed. The results revealed genetic differentiation among the populations in these different regions. They indicated substantial polymorphism in each population, with notable diversity in the number of alleles (18–52) and polymorphic information content (PIC, 0.78–0.93) across microsatellite loci. Low Fis values indicated limited inbreeding or genetic drift in Salalah and Masirah. FST values greater than 0.1 suggested significant differentiation of the Salalah population from Khasab and Suhar and low connectivity. Finally, we characterized the genetic diversity of five A. spinifer populations across the Arabian Gulf, the Sea of Oman, and the Arabian Sea using mitochondrial D-loop (control region). Three hundred samples from five locations were analyzed, revealing 66 haplotypes and high haplotype diversity detected through genetic diversity indices. Pairwise genetic differences indicated varying levels of population differentiation among the A. spinifer populations in Oman. The haplotypic network suggests complex genetic relationships, hinting at some migrations and potential localized adaptations, which signifies the importance of genetic-based conservation management strategies in the future.