ABSTRACT: Rainbow smelt Osmerus mordax uses the maximum turbidity zone (MTZ) of the St. Lawrence Estuary as a nursery area, with its prey field dominated by the calanoid copepod complex Eurytemora affinis. This cryptic species complex is a vital link between primary production and higher trophic levels, serving as crucial prey for fish larvae. We evaluated the contribution of the North-Atlantic clade (NAC) of E. affinis and the Atlantic clade (now described as E. carolleeae) to the diet of early stages of rainbow smelt across MTZ habitats. In summer 2021, 4 surveys revealed a heterogeneous distribution of smelt and its copepod prey with highest mean abundances found in the oligohaline habitat (0.5–5.0 PSU; 20 ± 15 larvae 100 m-3, 95% CI) compared to the limnetic (0–5.0 PSU; 5 ± 3 larvae 100 m-3, 95% CI) and mesohaline habitats (5.0–18.0 PSU; 11 ± 6 larvae 100 m-3, 95% CI). Ontogenetic diet shifts were observed, the E. affinis complex being the predominant prey in June and July (76–93% PSIRI), while the mysid Neomysis americana increased in importance in August (80% PSIRI, oligohaline habitat). Using a newly developed qPCR assay, we revealed that E. affinis NAC was the predominant prey with 72% of stomachs containing only this Eurytemora species; thus E. carolleeae played a minor role in energy transfer. Similarly, E. affinis NAC dominated in the environment, indicating rainbow smelt larvae exploit the most abundant resource available. This study highlights the critical role of E. affinis NAC in sustaining energy transfer pathways towards larval rainbow smelt for 1 year; however, interannual variation within the cryptic complex in the MTZ should be further assessed.