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Chaetoceros wighamii Brightwell is a planktonic diatom species originally described from brackish waters. Since its original description, the species has been reported in a wide variety of habitats, ranging from freshwater to marine. Varying descriptions exist in the taxonomic literature and several taxa have been considered as synonyms, including freshwater species Chaetoceros amanita. In this study we provide morphological and ultrastructural information on a cultured strain isolated from freshwater sample collected in the Lake Vrana (Vransko jezero) in Croatia, in April 2011. The cells form short and robust chains with very narrow apertures, often partially occluded by silica membranes. Other distinctive features observable in light microscopy are the shape and orientation of the setae which are very long, straight and robust, diverging in various directions from the chain axis and the single parietal chloroplast extending from valve to valve. Distinct ultrastructural characteristics are the absence of processes either in intercalary or terminal valves and the ornamentation of the valve face with densely distributed ribs spreading from an irregular eccentric hyaline area without a clearly defined annulus. The outer surface of the terminal valve is ornamented with small spines and setae are composed of flat longitudinal filaments interconnected with short bars and ornamented with small spines tightly arranged around the setae. Our description agrees well with that reported for the freshwater morphotypes of C. wighamii (syn. C. amanita) and contributes for a reliable distinction of this intriguing taxon from similar morphotypes. This finding supports the interpretation of Chaetoceros wighamii as a freshwater/brackish species and represents the first report of a Chaetoceros species in lacustrine environment in Croatia and possibly in any Central European habitats.