Identifying spawning sites for broadcast spawning fish species is a key element of delineating critical habitat for managing and regulating marine fisheries. Genetic barcoding has enabled accurate taxonomic identification of individual fish eggs, overcoming limitations of morphological classification techniques. In this study, planktonic fish eggs were collected at 23 stations along the northwestern coast of Cuba and across the Florida Straits to United States waters. A total of 564 fish eggs were successfully identified to 89 taxa within 30 families, with the majority of taxa resolved to species. We provide new spawning information for Luvarus imperialis (Louvar), Bothus lunatus (Plate Fish), Eumegistus illustris (Brilliant Pomfret), and many economically important species. Data from most sites supported previously established patterns of eggs from neritic fish species being found on continental shelves and oceanic species spawning over deeper waters. However, some sites deviated from this pattern, with eggs from reef‐associated fish species detected in the deep waters of the Florida Straits and pelagic species detected in the shallow, continental shelf waters off the coast of northwestern Cuba. Further investigation using satellite imagery revealed the presence of a mesoscale cyclonic eddy that likely entrained neritic fish eggs and transported them into the Florida Straits. The technique of combining DNA‐based fish egg identification with remotely‐sensed hydrodynamics provides an important new tool for assessing the interplay of regional oceanography with fish spawning strategies.