Benthic cyanobacterial Microcoleus spp. are known for their widespread geographic distribution, and can form thick benthic mats that are sometimes extremely toxic, due to the production of the potent neurotoxin, anatoxin-a. The mats often comprise anatoxin-producing (toxic) and non-anatoxin-producing (non-toxic) strains with varying relative abundance that determines mat toxicity. To examine the fundamental genomic differences between toxic and non-toxic strains, we sequenced eleven Microcoleus enrichment cultures and compared these to twenty-eight metagenome-derived Microcoleus genomes and three non-axenic culture assemblies.