Two of the remaining coregonine species in Lake Ontario, cisco (Coregonus artedi) and lake whitefish (C. clupeaformis), spawn in Chaumont Bay, NY. Larvae co-occur in the spring but are difficult to distinguish morphologically. We applied genetic species identification using microsatellite DNA loci of 268 larvae from known locations in nearshore and offshore habitats in Chaumont Bay to determine the extent of mixing of these species in each habitat. Cisco dominated (95% of larvae) the larvae in offshore habitats and lake whitefish dominated (84%) in nearshore habitats, where seven of eight putative hybrids occurred. Habitat segregation between these two species at the larval stage has implications for productivity estimates. Discrimination between cisco and lake whitefish larvae helps to characterize habitat and basic life history needs and to focus research collections. Genetic species identification should be applied to larger samples of larvae to evaluate changes in larval distributions and associations with environmental conditions.