Abstract
Freshwater fish biodiversity is undergoing a rapid decline worldwide due to habitat fragmentation, climate change, invasive species, overexploitation and pollution. Yet, for many regions, including biodiversity‐rich but underrepresented areas such as Türkiye, large‐scale assessments of species diversity and distribution remain limited. Environmental DNA (eDNA) metabarcoding offers a powerful, non‐invasive and scalable tool for biodiversity assessment, capable of detecting both common and cryptic taxa. Here, we present the first nationwide, basin‐scale eDNA metabarcoding survey of freshwater fishes across Türkiye's major river systems, spanning a transect of more than 6000 km and encompassing 29 sampling sites across seven principal basins. Triplicate water samples were analysed using high‐throughput sequencing of the mitochondrial 12S ribosomal RNA (rRNA) gene, generating 351,392 high‐quality reads (after filtration) and detecting 52 fish species, including native, invasive and several taxa previously unrecorded in their respective basins. The Eastern Mediterranean basin exhibited the highest species richness (28 species) and diversity ( H ′ = 2.35; 1 – D = 0.88), whereas beta‐diversity analyses revealed marked spatial structuring among basins (Jaccard similarity 0.17–0.58) and a clear biogeographic separation in principal co‐ordinates analysis (PCoA) ordination. Importantly, eDNA metabarcoding uncovered cryptic and low‐abundance taxa not detected by conventional surveys, demonstrating its complementary value and sensitivity. By providing the first comprehensive molecular baseline of Turkish freshwater ichthyofauna, this study illustrates the potential of large‐scale eDNA approaches to transform biodiversity monitoring, inform conservation strategies and support management decisions in freshwater ecosystems facing accelerating environmental change.