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South Florida (USA) has a subtropical to tropical climate with an extensive and diverse coastline that supports the growth of benthic cyanobacterial mats (BCMs). These BCMs are widespread and potentially house numerous bioactive compounds; however, the extent of the cyanobacterial diversity within these mats remains largely unknown. To elucidate this diversity, BCMs from select locations in South Florida were sampled and isolated into unicyanobacterial cultures for morphological and molecular studies. Phylogenetic relationships of isolated taxa were assessed using the markers 16S rRNA and 16S‐23S rRNA ITS by both maximum likelihood and Bayesian inference. We propose Affixifilum gen. nov. based on morphological characteristics and the 16S rRNA phylogeny. Two species are included: Affixifilum granulosum comb nov. (=Neolyngbya granulosa) found in Brazil and Florida (USA) and A. floridanum sp. nov. Several other features, including pair‐wise distance of 16S rRNA and 16S‐23S rRNA ITS, 16S‐23S rRNA ITS secondary structure, morphology, and ecology, provide support for Affixifilum. We also propose the transfer of Lyngbya regalis to Neolyngbya as N. regalis comb. nov. and include the description of one novel species, N. biscaynensis sp. nov.