Veríssimo, JoanaLopes‐Lima, ManuelAmaral, FábioChaves, CátiaFernandes, VascoKemanja, MutaleniTeixeira, AmilcarMartins, Filipa M. S.Beja, Pedro2025-07-212025-07-212025Veríssimo, Joana; Lopes‐Lima, Manuel; Amaral, Fábio; Chaves, Cátia; Fernandes, Vasco; Kemanja, Mutaleni; Teixeira, Amilcar; Martins, Filipa M. S.; Beja, Pedro. (2025). Navigating Methodological Trade‐Offs in eDNA Metabarcoding Biodiversity Monitoring: Insights From a Mediterranean Watershed. Molecular Ecology Resources. ISSN 1755-098X. 25:6, p, 1-141755-098Xhttp://hdl.handle.net/10198/34680Environmental DNA (eDNA) metabarcoding technologies promise significant advances in biodiversity monitoring, yet their application requires extensive optimisation and standardisation. Recent research demonstrated that increased sampling and analytical efforts are needed to improve biodiversity estimates, though fully optimising study designs is often hindered by resource constraints. Consequently, researchers must carefully navigate methodological trade‐offs to design effective eDNA metabarcoding monitoring studies. We conducted a water eDNA survey of vertebrates in a Mediterranean watershed to identify key methodological factors influencing species richness and composition estimates. We examined the impacts of using high‐ versus low‐capacity filtration capsules, varying levels of biological and technical replication, and the pooling of PCR replicates before indexing. The primary sources of variation identified were capsule filtration capacity and site replication across the watershed. While biological replication within sites and PCR replication also improved biodiversity estimates, their effects were comparatively smaller. Pooling PCR replicates before indexing performed more poorly than analysing them independently. Methodological impacts were stronger on terrestrial than on aquatic species. Based on these results, we recommend that priority should be given to high‐capacity filtration and sampling across multiple sites. Site‐level replication deserves lower priority, especially when filtering large water volumes. PCR replication is crucial for detecting rare species but should be balanced with increased site sampling and eventually site‐level replication. Avoiding the pooling of PCR replicates is important to enhance sensitivity for rare species. Overall, we stress the importance of balancing methodological choices with resource constraints and monitoring goals, and we emphasise the need for research assessing methodological trade‐offs in different study systems.engBiodiversity monitoringEnvironmental DNAFreshwater ecosystemsHigh-throughput sequencingMetabarcodingReplicationjournal article10.1111/1755-0998.140821755-0998