This paper focuses on the morphology, taxonomy and ecology of the widespread cyanobacteria of the genus Nodularia Mertens ex Bornet & Flahault. In this study the benthic strain of N. sphaerocarpa, isolated from a sand-pit near Olomouc (Czech Republic), is compared with brackish and seawater strains. Changes in morphology and growth parameters (biomass and chlorophyll a) recorded in varying salinity gradients were studied and a 16S rRNA sequencing and AFLP analysis conducted. Morphological and ecophysiological characteristics found were in congruence with molecular data. Three major subgroups of the benthic Nodularia (N. sphaerocarpa, N. moravica and N. harveyana) were found using the polyphasic approach. The results of both the molecular and morphological study clearly separated N. moravica and N. sphaerocarpa, as freshwater species preferring a low salinity and the N. harveyana strains originating from a marine environment preferring a high salinity.