The genus Esoptrodinium Javornický consists of freshwater, athecate dinoflagellates with an incomplete cingulum. Strains isolated thus far feed on microalgae and most possess obvious pigmented chloroplasts, suggesting mixotrophy. However, some geographic isolates lack obvious pigmented chloroplasts. The purpose of this study was to comparatively examine this difference and the associated potential for mixotrophy among different isolates of Esoptrodinium. All isolates phagocytized prey cells through an unusual hatch-like peduncle located on the ventral episome, and were capable of ingesting various protist taxa. All Esoptrodinium isolates required both food and light to grow. However, only the tested strain with visible pigmented chloroplasts benefited from light in terms of increased biomass (phototrophy). Isolates lacking obvious chloroplasts received no biomass benefit from light, but nevertheless required light for sustained growth (i.e., photoobligate, but not phototrophic). Isolates with visible chloroplasts exhibited chlorophyll autofluorescence and formed a monophyletic psbA gene clade that suggested Esoptrodinium possesses inherited, peridinoid-type plastids. One isolate with cryptic, barely visible plastids lacked detectable chlorophyll and exhibited an apparent loss-of-function mutation in psbA, indicating the presence of nonphotosynthetic plastids. The other isolate that lacked visible chloroplasts lacked both detectable chlorophyll and an amplifiable psbA sequence. The results demonstrate mixotrophy quantitatively for the first time in a freshwater dinoflagellate, as well as apparent within-clade loss of phototrophy along with a correlated mutation sufficient to explain that phenotype. Phototrophy is a variable trait in Esoptrodinium; further study is required to determine if this represents an inter- or intraspecific (allelic) characteristic in this taxon.