Benthic cyanobacteria, particularly Microcoleus, represent one of the most significant types of harmful algal blooms due to their increasing prevalence and profound environmental and animal health impacts. In May 2023, benthic cyanobacteria dominated by Microcoleus were observed in a small tributary of the Virgin River in Zion National Park, Utah. Samples were collected from six sites: three on rock substrates and three on sand. Paired benthic mat and downstream water samples were analyzed to investigate ecological dynamics. Metagenomic analysis revealed that cyanobacteria constituted over 60% of benthic mats, primarily from the Microcoleus genus, with less than 5% of sequences attributed to eukaryotic algae (see Figure 1). Toxin analysis by LC-MS/MS detected anatoxin-a (ATX) (377.13 ± 18.05 µg/g) and dihydroanatoxin-a (5.15 ± 0.3 µg/g) in all benthic mats, while ATX (0.377 µg/L) was also present in water. No homoanatoxin-a or its derivatives were detected. Low chlorophyll-a levels and microscopy suggested benthic, not pelagic, sources of toxins. Resampling in fall 2023 revealed a complete shift in cyanobacterial composition, with Microcoleus absent and no ATX detected in mats or water. Distinct Microcoleus monoclonal strains, isolated in April 2023 under study to compare genotypic differences between laboratory-cultured and environmental samples. This study employs metagenomics and gene expression analysis to explore Microcoleus survival mechanisms in oligotrophic environments, interactions with other microbial taxa, and substrate influences on benthic mat growth and toxin production. These findings provide insights into ecological drivers of harmful algal bloom formation in freshwater systems