We investigated the distribution of bioluminescent dinoflagellates in the Patagonian Shelf region using “universal” PCR primers for the dinoflagellate luciferase gene. Luciferase gene sequences and single cell PCR tests, in conjunction with taxonomic identification by microscopy, allowed us to identify and quantify bioluminescent dinoflagellates. We compared these data to coincidental discrete optical measurements of stimulable bioluminescence intensity. Molecular detection of the luciferase gene showed that bioluminescent dinoflagellates were widespread across the majority of the Patagonian Shelf region. Their presence was comparatively underestimated by optical bioluminescence measurements, whose magnitude was affected by interspecific differences in bioluminescence intensity and by the presence of other bioluminescent organisms. Molecular and microscopy data showed that the complex hydrography of the area played an important role in determining the distribution and composition of dinoflagellate populations. Dinoflagellates were absent south of the Falkland Islands where the cold, nutrient-rich, and well-mixed waters of the Falklands Current favoured diatoms instead. Diverse populations of dinoflagellates were present in the warmer, more stratified waters of the Patagonian Shelf and Falklands Current as it warmed northwards. Here, the dinoflagellate population composition could be related to distinct water masses. Our results provide new insight into the prevalence of bioluminescent dinoflagellates in Patagonian Shelf waters and demonstrate that a molecular approach to the detection of bioluminescent dinoflagellates in natural waters is a promising tool for ecological studies of these organisms.