The use of environmental DNA (eDNA) is a relatively new tool in New Zealand, and its increased use worldwide in the last decade has seen a surge in its utility and application. The information gaps in its use and effectiveness in the field of environmental science are fast being explored and answered. However, many questions remain unanswered, particularly in an Aotearoa New Zealand context. The aim of this thesis is to extend the knowledge needed for effective and efficient use of this new and exciting tool in lotic Aotearoa New Zealand conditions. National standardisation of ecological sampling protocols between different agencies in Aotearoa New Zealand has historically been difficult to attain, creating challenges for combining datasets for national scale analyses. The introduction of new methods for biological monitoring, such as environmental DNA (eDNA), presents an opportunity to standardise aquatic sampling protocols prior to widespread adoption. The objective of this study (chapter two) was to optimise eDNA sample replication for the consistent characterisation of freshwater fish and macroinvertebrate communities in flowing waters, and ultimately, to inform the development of robust national monitoring standards. A comparison of field replication and extraction methods (pooling of preservation buffer) was also trialled as part of this high replication (n=16) eDNA study to assess any potential benefits in measuring species richness and reducing processing costs alongside replication optimisation. This involved two ‘syringe’ sampling methods (‘standard’ and ‘boosted’, eight each) conducted across 54 riverine sites throughout the country. No significant difference was found for species richness between the standardised (eight replicates) or boosted (16 replicates composited to eight) eDNA methods for fish and macroinvertebrates. Results indicated that six replicates were needed to consistently detect 89.5% of fish species likely to be present using field-based syringe eDNA sampling and preservation. However, an altitudinal species richness effect was observed for fish. For macroinvertebrates, six replicates were required to identify 86% of taxa identified to the NEMS (National Environmental Monitoring Standards) level used for the Macroinvertebrate Community Index (MCI: usually genera) while eight replicates were required to detect 89% of NEMS taxa. For fish and macroinvertebrate biodiversity, this study suggests that six replicates are a reasonable trade-off between effective community characterisation and cost in Aotearoa New Zealand lotic systems. Understanding the spatial limits and the way in which eDNA behaves in lotic systems is important to understand when designing eDNA sampling programmes and interpreting results. Questions around biodiversity detection at spatiotemporal scales in relation to stream order and the influence of tributaries remain. We sought to extend our knowledge of longitudinal eDNA dispersal and detectability in wadable lotic systems by using live caged fish and a naturally occurring habitat specific fish species. An additional unexpected external molecular source was also included in the analysis. An eDNA collection method comparison between active and passive sampling techniques was also trialled alongside caged experiments in the second half of this study. A correlation between stream discharge and the distance in which eDNA travels downstream is reported. An initial drop in eDNA concentration from caged fish over the first kilometre downstream is followed by a peak 4 km downstream, is suggested to be reflective of plume dynamics. Tributary-specific fish species at low biomass and their associated eDNA signal is largely diluted and not detectable directly downstream of confluences, though there was some evidence that if sampled further downstream in the mainstem, tributary signals may be detected. The caged fish trials showed how far an eDNA signal can travel, while results from naturally occurring longitudinal signals confirm that eDNA metabarcoding can measure biodiversity at spatiotemporal scales. Active sampling in general outperformed passive sampling. Issues around field contamination and sampling strategies based on results from this study are also discussed. Overall, this thesis provides insights and potential guidance in the use of eDNA in lotic Aotearoa New Zealand systems. It highlights the importance of eDNA field replication in the measurement of biodiversity for fish and macroinvertebrates and some terrestrial species. It helps interpret the ecology of eDNA in distance and structure. This information may assist in future eDNA sampling strategies and monitoring.