Estuaries are one of the most ecologically and economically valuable ecosystems on the planet, they are also among the most degraded. Currently, in the United Kingdom the ecological quality of estuaries (‘transitional waters’) is monitored by assessments of key ecological groups, such as fish, to meet the demands of legislation derived from the Water Framework Directive (WFD). At present within Wales and England, Natural Resources Wales (NRW) and the Environment Agency (EA), respectively, use several combined fishing methods to survey fish communities. Equivalent methodologies are used across Europe. However, multimethod fishing is expensive, difficult to implement consistently and potentially destructive. Environmental DNA (eDNA) analysis technology is an emerging survey method for fish. There are still relatively few studies using eDNA metabarcoding to assess fish biodiversity in estuaries. The aim of this project is to conduct the basic research which will enable the future development of an eDNA metabarcoding tool to assess the biodiversity of fishes in estuaries. Three studies were conducted in macrotidal estuaries in Wales and England. Comparable methodologies were used to generate data on fish eDNA. Replicate surface water samples were collected at each sampling event/station, DNA was extracted from samples and subjected to metabarcoding analysis using an assay targeting teleost fishes. Fish assemblage data was analysed using up to date statistical approaches. The first study aimed to compare the spatial fish assemblage composition detected via eDNA with conventional fishing surveys, and also investigate seasonal patterns, in the Dee (Wales). Sampling was conducted alongside three fishing gear types, in a spatially systematic design in October 2018. Seasonal changes in composition were investigated by re-sampling a subset of stations using eDNA only, in June 2018. In autumn, eDNA detected the majority of species caught by fishing, and detected a greater species richness, per a given sampling effort, than two gear types. Assemblage composition was also correlated with salinity, consistently across seasons. The aim of the second study was to investigate the short-term variability in the fish assemblage, in the Conwy Estuary (Wales). In Autumn 2020, samples were taken at a single station at high and low tide over 15 days, covering a spring to neap tidal cycle. Temporal variation in the assemblage composition of fish species were correlated with changes in salinity, which occurred at different tidal states and due to an episodic increase in river flow, and to a lesser extent tidal range. The third study aimed to compare the fish assemblage detected via eDNA to fishing gears in three estuaries in northeast England, over two seasons: early summer and autumn. Environmental DNA was sampled alongside fishing at multiple sampling stations in two estuaries in autumn 2016, and in all three estuaries in early summer and autumn 2017. The majority of species caught by fishing were detected by eDNA, including species of conservation interest and a none-native. Species richness estimates for each estuary were in some cases greater using eDNA compared to fishing. Numerous novel species were detected via eDNA and a different assemblage composition was detected relative to one netting type. Analyses of eDNA separately from fishing showed it could detect differences in assemblage composition between seasons and estuaries. In conclusion, eDNA may be an effective method to survey fishes in estuaries for biomonitoring purposes. Correlations in the eDNA assemblage with temporal and spatial variation in ecological variables, such as salinity, also have important implications for biomonitoring survey design. The implications of these studies for biomonitoring, and the requirements for further research are discussed throughout and summarised in the general discussion.