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Cyanobacterial harmful algal proliferations (cyanoHAPs) are increasingly associated with dog and livestock deaths when benthic mats break free of their substrate and float to the surface. Fatalities have been linked to neurotoxicosis from anatoxins, potent alkaloids produced by certain genera of filamentous cyanobacteria. After numerous reports of dog illnesses and deaths at a popular recreation site on Lady Bird Lake, Austin, Texas in late summer 2019, water and floating mat samples were collected from several sites along the reservoir. Water quality parameters were measured and mat samples were maintained for algal isolation and DNA identification. Samples were also analyzed for cyanobacterial toxins using LC-MS. Dihydroanatoxin-a was detected in mat materials from two of the four sites (0.6–133 ng/g wet weight) while water samples remained toxin-free over the course of the sampling period; no other cyanobacterial toxins were detected. DNA sequencing analysis of cyanobacterial isolates yielded a total of 11 genera, including Geitlerinema, Tyconema, Pseudanabaena, and Phormidium/Microcoleus, taxa known to produce anatoxins, including dihydroanatoxin, among other cyanotoxins. Analyses indicate that low daily upriver dam discharge, higher TP and NO3 concentrations, and day of the year were the main parameters associated with the presence of toxic floating cyanobacterial mats.