The genera Esoptrodinium Javornický and Bernardinium Chodat comprise freshwater, athecate dinoflagellates with an incomplete cingulum but differing reports regarding cingulum orientation and the presence of chloroplasts and an eyespot. To examine this reported diversity, six isolates were collected from different freshwater ponds and brought into clonal culture. The isolates were examined using LM to determine major cytological differences, and rDNA sequences were compared to determine relatedness and overall phylogenetic position within the dinoflagellates. All isolates were athecate with a left-oriented cingulum that did not fully encircle the cell, corresponding to the current taxonomic concept of Esoptrodinium. However, consistent cytological differences were observed among clonal isolates. Most isolates exhibited unambiguous pale green chloroplasts and a distinct bright-red eyespot located at the base of the longitudinal flagellum. However, one isolate had cryptic chloroplasts that were difficult to observe using LM, and another had an eyespot that was so reduced as to be almost undetectable. Another isolate lacked visible chloroplasts but did possess the characteristic eyespot. Nuclear rDNA phylogenies strongly supported a monophyletic Esoptrodinium clade containing all isolates from this study together with a previous sequence from Portugal, within the Tovelliaceae. Esoptrodinium subclades were largely correlated with cytological differences, and the data suggested that independent chloroplast and eyespot reduction and/or loss may have occurred within this taxon. Overall, the isolates encompassed the majority of cytological diversity reported in previous observations of Bernardinium/Esoptrodinium in field samples. Systematic issues with the current taxonomic distinction between Bernardinium and Esoptrodinium are discussed.