Environmental DNA (eDNA) metabarcoding has revolutionized biomonitoring in both marine and freshwater ecosystems. However, for semi‐aquatic and terrestrial animals, the application of this technique remains relatively untested. We first assess the efficiency of eDNA metabarcoding in detecting semi‐aquatic and terrestrial mammals in natural lotic ecosystems in the UK by comparing sequence data recovered from water and sediment samples to the mammalian communities expected from historical data. Secondly, using occupancy modelling we compared the detection efficiency of eDNA metabarcoding to multiple conventional non‐invasive survey methods (latrine surveys and camera trapping). eDNA metabarcoding detected a large proportion of the expected mammalian community within each area. Common species in the areas were detected at the majority of sites. Several key species of conservation concern in the UK were detected by eDNA sampling in areas where authenticated records do not currently exist, but potential false positives were also identified. Water‐based eDNA metabarcoding provided comparable results to conventional survey methods in per unit of survey effort for three species (water vole, field vole and red deer) using occupancy models. The comparison between survey ‘effort’ to reach a detection probability of ≥.95 revealed that 3–6 water replicates would be equivalent to 3–5 latrine surveys and 5–30 weeks of single camera deployment, depending on the species. Synthesis and applications. eDNA metabarcoding can be used to generate an initial ‘distribution map’ of mammalian diversity at the landscape level. If conducted during times of peak abundance, carefully chosen sampling points along multiple river courses provide a reliable snapshot of the species that are present in a catchment area. In order to fully capture solitary, rare and invasive species, we would currently recommend the use of eDNA metabarcoding alongside other non‐invasive surveying methods (i.e. camera traps) to maximize monitoring efforts.

1.Environmental DNA (eDNA) metabarcoding has revolutionised biomonitoring in both marine and freshwater ecosystems. However, for semi-aquatic and terrestrial animals, the application of this technique remains relatively untested. 2.We first assess the efficiency of eDNA metabarcoding in detecting semi-aquatic and terrestrial mammals in natural lotic ecosystems in the UK by comparing sequence data recovered from water and sediment samples to the mammalian communities expected from historical data. Secondly, we evaluate the detection efficiency of eDNA samples compared to multiple conventional non-invasive survey methods (latrine surveys and camera trapping) using occupancy modelling.3. eDNA metabarcoding detected a large proportion of the expected mammalian community within each area. Common species in the areas were detected at the majority of sites. Several key species of conservation concern in the UK were detected by eDNA in areas where authenticated records do not currently exist, but potential false positives were also identified for several non-native species. 4. Water-based eDNA samples provided comparable results to conventional survey methods in per unit of survey effort for three species (water vole, field vole, and red deer) using occupancy models. The comparison between survey 'effort' to reach a detection probability of ≥ 0.95 revealed that 3-6 water replicates would be equivalent to 3-5 latrine surveys and 5-30 weeks of single camera deployment, depending on the species. Synthesis and Applications. eDNA metabarcoding represents an extremelypromising tool for monitoring mammals, allowing for the detection of multiple species simultaneously, and provides comparable results to widely-used conventional survey methods. eDNA from freshwater systems delivers a 'terrestrial dividend' by detecting both semi-aquatic and terrestrial mammalian communities, and provides a basis for future monitoring at a landscape level over larger spatial and temporal scales (i.e. long-term monitoring at national levels). modelling species identified by metabarcoding are rare (Abrams et al., 2019; Valentini et al., 2016).The aim of this study was to assess the efficiency of eDNA for detecting semiaquatic and terrestrial mammals in natural lotic systems in the UK. We conducted eDNA sampling in rivers and streams in two areas (Assynt, Scotland and Peak District National Park, England), which together have the majority of UK semi-aquatic and terrestrial mammalian species present (Table S1). Our objectives were two-fold: first, we sought to establish whether eDNA metabarcoding is a viable technique for monitoring semi-aquatic and terrestrial mammals by comparing it to the mammalian communities expected from historical data, a group for which eDNA sampling has rarely been evaluated in a natural setting. Secondly, we evaluate the detection efficiency of water-and sediment-based eDNA sampling in one of these areas (Assynt) for multiple species compared to multiple conventional non-invasive survey methods (latrine surveys and camera ...