Cisco (Coregonus artedi) and lake whitefish (Coregonus clupeaformis) are native fish species of management concern in the Laurentian Great Lakes that often overlap in spawning locations and timing. Thus, species-level inference from in situ sampling requires methods to differentiate their eggs. Genetic barcoding and hatching eggs to visually identify larvae are used but can be time and cost intensive. Observations in published literature indicate that lake whitefish eggs may be larger than cisco eggs in the Great Lakes, but this has not yet been substantiated. Samples from shared spawning grounds are unlikely to contain similarly sized or colored eggs from other species. Thus, we assessed whether lake whitefish and cisco eggs could be separated based on size alone. Fertilized, hardened eggs were collected in situ during spawning at Elk Rapids, Lake Michigan and Chaumont Bay, Lake Ontario and preserved in ethanol. Individual eggs were measured and genetically identified. Mean diameter for cisco (2.45 mm, SD = 0.22, n = 444) was smaller than for lake whitefish (3.21 mm, SD = 0.20, n = 99). We used classification trees to identify a species-separating size threshold of 2.88 mm (95% bootstrap CI = [2.877, 2.976]), which classified eggs with an accuracy rate of 96%. Differences between species across other samples from the same locations were mostly consistent with the threshold size, but we suggest validation if applying this method to other populations. Separation of cisco and lake whitefish eggs by diameter can be accurate, efficient, and especially suitable for large sample sizes.