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Abstract Illegal trade in sharks and rays continues to undermine global conservation efforts, with enforcement often hampered by the inability to identify products to the species level. Here, we present a portable, cost-effective High-Resolution melt (HRM) assay for rapid DNA-based identification of elasmobranch species in trade. Using a reference library of 669 vouchered tissue samples collected from field operations and international market surveys, we validated the assay’s capacity to accurately differentiate at least 55 shark and ray species based on melt curve profiles, including 38 species listed under the Convention on International Trade in Endangered Species of Wild Fauna and Flora. Automated image classification enabled high-throughput identification with 99.2% accuracy. The assay yields results within two hours at a per-sample cost of $1.50, and is compatible with portable qPCR platforms, making it suitable for on-site applications. This approach represents a scalable molecular enforcement tool that can empower local authorities to monitor trade more effectively, support compliance with international regulations, and enhance global efforts to combat wildlife trafficking.