Native to the central USA, the Blue Catfish Ictalurus furcatus and the Flathead Catfish Pylodictis olivaris have been widely introduced into many Atlantic slope rivers and are now found in several drainages of the Chesapeake Bay. Fisheries managers are concerned that these large, long‐lived catfish species may be contributing to observed declines in anadromous species, such as the American Shad Alosa sapidissima, Blueback Herring A. aestivalis, and Alewife A. pseudoharengus, all of which once comprised major U.S. fisheries. We assessed spatiotemporal variability and selectivity in the diets of Blue Catfish and Flathead Catfish during the spawning migration of these alosines. Catfish stomachs were collected during March–May in nontidal freshwater, tidal freshwater, oligohaline, and mesohaline portions of the James River. Diet contents were extracted from 2,495 catfish, 69.86% of which had prey items present in their foreguts (N = 1,743). We used DNA barcoding to identify degraded fish prey; nearly 30 taxa that would have otherwise gone undetected were identified in this manner. Blue Catfish had broad, omnivorous diets, whereas Flathead Catfish fed solely on other fish. Alosines were found in 4.46% of Blue Catfish stomachs and 16.67% of Flathead Catfish stomachs. Flathead Catfish selectively preyed on American Shad and, to a lesser degree, river herring. Alosines were consumed more frequently in nontidal freshwater areas, particularly in the high‐gradient reach between Bosher Dam and the 14th Street Bridge, and predation on alosines peaked in April. Our results suggest that Flathead Catfish are likely to have a greater per capita impact on depleted alosines. Furthermore, dams and other obstacles to fish movement may increase alosines’ vulnerability to predation by large catfish.