The presence of toxigenic benthic cyanobacteria in riverine ecosystems is an increasing concern around the world. In 2018, the death of three dogs along the Wolastoq (also known as the Saint John River) in New Brunswick, Canada, was attributed to anatoxin exposure after they ingested benthic microbial mats found along the shore. Here, we shotgun sequenced the DNA of 15 non-axenic cyanobacterial isolates derived from four anatoxin-containing benthic mat samples associated with the dog deaths. Anatoxins were produced by some of the isolates, but not all. We retrieved near-complete Microcoleus metagenome-assembled genomes (MAGs) from the isolates that are closely related to anatoxin-producing Microcoleus from the Cardrona River (New Zealand), although the Microcoleus MAGs from the Wolastoq varied in the presence/absence of the anatoxin-a biosynthesis cluster. Sequence similarity at the genomic level suggests that toxigenic and non-toxigenic Microcoleus MAGs from the Wolastoq belong to the same species but are separate subspecies. The toxigenic and nontoxic Wolastoq Microcoleus subspecies coexisted in the mat samples in similar relative abundance. Overall genomic comparisons revealed that toxigenic Microcoleus MAGs are longer and code for more accessory genes than their non-toxigenic relatives, suggesting a differential responsiveness to changing environments, stress conditions and nutrient availability.