Sea lamprey (Petromyzon marinus) are invasive in the Laurentian Great Lakes, parasitize large-bodied fishes, and therefore are the focus of an international control program. However, damage caused by sea lamprey to modern day fish stocks remains uncertain because diet analysis of juvenile sea lamprey has been challenging; they feed on blood and are difficult to randomly sample in the lakes. Here, both challenges were addressed by showing that DNA metabarcoding of fecal material can be used to identify the diet of actively feeding juvenile sea lamprey, and can also be used to determine what non-feeding adult sea lamprey captured in streams fed on while parasitizing fish. Fecal samples from juvenile sea lamprey that were feeding on lake trout in northern Lake Huron overwhelmingly contained lake trout (Salvelinus namaycush) DNA (90%), while smaller percentages contained lake whitefish (Coregonus clupeaformis; 5%) and longnose sucker (Catostomus catostomus; 5%) DNA. Fecal samples from adult sea lamprey captured from a tributary to northern Lake Huron overwhelmingly contained longnose and white sucker DNA (Catostomus spp.; 80%), while a smaller percentage contained lake trout DNA (10%). Diet composition of adult sea lamprey sampled in the tributary (Black Mallard Creek) was more diverse than juvenile diet composition. DNA metabarcoding suggests that Catostomus spp. may be an important host fish in northern Lake Huron for sea lamprey prior to spawning. Future research could investigate how diet varies across years and lakes and the prevalence and sources of DNA contamination. Application of DNA metabarcoding for diet assessment may be practical for identifying populations of invasive sea lamprey that feed on highly valued fishes and help guide restoration of lampreys worldwide.