The spotted eagle ray Aetobatus ocellatus (Kuhl, 1923) has a widespread Indo-West Pacific distribution and displays substantial population genetic structuring. Genetic data are crucial for understanding the species’ diversity, connectivity, and adaptation. However, molecular genetic information on A. ocellatus from Melanesia is lacking, which impedes our understanding of gene flow among geographic regions. In this study, we sampled 45 A. ocellatus, primarily from Fiji’s largest fish market in the capital, Suva. Mitochondrial DNA Cytochrome C Oxidase subunit I (COI) barcoding was used for species identification, and DArT-seqTM technology was applied to assess the nuclear genetic diversity. Barcoding of the COI gene showed a 98.6% to 99.8% similarity to A. ocellatus reference sequences in the Barcode of Life Data System, and the 45 individuals were represented by three major evolutionary haplotype clusters. Genotyping resulted in 24,313 Single-Nucleotide Polymorphisms (SNPs) which were quality-filtered to 7094 SNPs per individual. The observed heterozygosity level was 0.310. The inbreeding coefficient was positive, and genotyping identified one full-sibling pair and one half-sibling pair from the 45 individuals. Additionally, eagle rays exhibit polychromatic patterns, and at least three ventral pattern variations were recorded in specimens from the market. Collectively, our main findings characterize the genetic profile of A. ocellatus in Fiji and can help to understand the diversification of this species within the region.