Cyanobacteria belonging to order Oscillatoriales can form large benthic mats in rivers, some of which produce the neurotoxin anatoxin-a. Mats of potentially toxic cyanobacteria are common, abundant, and diverse in both forks of the Shenandoah River, VA, USA but little is known about their taxonomies and toxicities. In 2023 and 2024, we collected benthic mats growing in the Shenandoah River and conducted integrative molecular, morphological, and toxin characterization of Microcoleus strains isolated from the samples. Using isolated filaments from field samples and monoclonal unialgal cultures, whole genome sequencing was successfully completed for three of the cyanobacterial species using either the IonTorrent S5 system or Illumina MiSeq systems to produce short reads which were de novo assembled into contigs. Phylogenetic analyses of the 16S rRNA gene identified two distinct species of Microcoleus, one non-toxic and one produced anatoxin-a and dihydroanatoxin-a. According to 16S phylogenetic analysis it is closely related to Microcoleus anatoxicus but the species identity is inconclusive. However, the most abundant mat-forming species in Shenandoah River, fit the morphological description of Microcoleus lacustris Farlow ex Gomont, but was phylogenetically distant from members of the Family Microcoleaceae. It was placed within the Family Coleofasciculaceae and appears to be a non-toxic undescribed member of the genus Limnofasciculus.