Benthic cyanobacterial mats in flowing waters are complex communities typically composed of taxa from the orders Coleofasciculales and Oscillatoriales, many of which have unresolved taxonomic positions and poorly characterized toxic potential. We collected field mats of a benthic non‐heterocytous filamentous cyanobacterium from the North and South Forks of the Shenandoah River in Northern Virginia (USA) that were dominated by a novel morphotype. Whole‐genome and 16S rRNA gene phylogenetic analyses placed this cyanobacterium within the recently described genus Limnofasciculus (Coleofasciculaceae). Genome‐based species delimitation metrics fell below accepted thresholds for bacterial species delineation relative to Limnofasciculus baicalensis, the only formally described species in the genus to date, supporting recognition of the cyanobacterium from Shenandoah River as a distinct species. Furthermore, the two Limnofasciculus species exhibited marked structural differences in the Box B helix of the 16S–23S ITS region. Comparative genomic analyses revealed a genome size similar to L. baicalensis and a conserved core gene repertoire alongside substantial divergence in biosynthetic gene cluster composition. Neither species contains biosynthetic gene clusters associated with the production of known cyanotoxins. Based on morphological, phylogenetic and genomic evidence, we describe Limnofasciculus delicatus sp. nov., supported by light microscopy and whole‐genome characterization.