AbstractCyanobacteria exhibit a vast diversity from polar to tropical environments. Though much work has been done on elucidating their biodiversity, knowledge on the occurrence, diversity and toxicity of benthic cyanobacteria is limited when compared to the planktonic forms. Integrating molecular techniques with ecological and morphological analyses has become essential in untangling cyanobacterial diversity, particularly for benthic taxa such as the cryptic “Lyngbya.” Molecular markers such as the 16S rRNA gene and whole genome sequencing have significantly improved the taxonomy of cyanobacteria. Building on these advancements, this study characterizes benthic cyanobacterial isolates from various locations in Florida, USA, and Orange Walk, Belize, resulting in the identification of a novel genus, Floridanema, and four new species (F. aerugineum, F. evergladense, F. flaviceps, and F. fluviatile). This new genus commonly occurs in canals, ponds, lakes and rivers. By integrating ecological, morphological, and genomic analyses, this study provides support for the family Aerosakkonemataceae and the establishment of the order Aerosakkonematales. The LC–MS data revealed that Floridanema strains do not produce microcystins, nodularin‐R, or anabaenopeptins.