ABSTRACTClostridium botulinumtype E toxin is responsible for extensive mortality of birds and fish in the Great Lakes. TheC. botulinum bontEgene that produces the type E toxin was amplified with quantitative PCR from 150 sloughed algal samples (primarilyCladophoraspecies) collected during summer 2012 from 10 Great Lakes beaches in five states; concurrently, 74 sediment and 37 water samples from four sites were also analyzed. ThebontEgene concentration in algae was significantly higher than in water and sediment (P< 0.05), suggesting that algal mats provide a better microenvironment forC. botulinum. ThebontEgene was detected most frequently in algae at Jeorse Park and Portage Lake Front beaches (Lake Michigan) and Bay City State Recreation Area beach on Saginaw Bay (Lake Huron), where 77, 100, and 83% of these algal samples contained thebontEgene, respectively. The highest concentration ofbontEwas detected at Bay City (1.98 × 105gene copies/ml of algae or 5.21 × 106g [dry weight]). This study revealed that thebontEgene is abundant in the Great Lakes but that it has spatial, temporal, and matrix variability. Further, embayed beaches, low wave height, low wind velocity, and greater average water temperature enhance thebontEoccurrence.