Proliferations of anatoxin-producing benthic cyanobacteria, notably Microcoleus, threaten aquatic ecosystems and public health. Our understanding of temporal variation in anatoxin concentrations within river systems has improved, but predicting when and where anatoxin concentrations are elevated across a watershed remains a significant challenge. To further understand the spatial scale of variation in anatoxins and their environmental drivers across a watershed, we sampled six mainstem and eight tributary locations distributed throughout the Scott River watershed in Northern California, USA.Four sites were previously managed for anatoxins by the Quartz Valley Natural Resource Office (QVIR) during 2022 and 2023. We chose sampling locations to span a wide range of stream sizes and watershed land use compositions to explore the relative influence of hypothesized drivers of Microcoleus proliferations, including nutrient and light availability, water temperature, specific conductance, stream discharge, and streambed substrate size. We sampled sites once a month from June to October of 2024 to capture the temporal variation within sites. At each site we measured environmental parameters expected to drive anatoxins, estimated percent cover of Microcoleus mats, and collected mat samples to quantify anatoxin concentrations and community composition. Microcoleus was documented across tributary and mainstem sites throughout the sampling period indicating that it is widely present in the Scott River watershed despite site differences. Early season mats were most extensive in tributaries but there was an overall increase in the percent of streambed covered by Microcoleus mats during August in tributaries and mainstem sites. Overall toxin levels were relatively low during our 2023 study compared to 2022. Improving our understanding of environmental drivers of Microcoleus mat distribution and anatoxin concentrations can further inform monitoring strategies to mitigate ecological and public health risks.