Largemouth (LMB: Micropterus salmoides) and Smallmouth Bass (SMB: Micropterus dolomieu) are important species in the recreational fisheries of the Laurentian Great Lakes. The invasion of the Round Goby (Neogobius melanostomus) into these lakes has changed several facets of black bass biology, but there is still much to learn about the relationship between these species. Previous dietary analyses have shown Round Goby to be important prey for bass, but have been limited by low visual identification rates of dissected stomach items. Within the present study, DNA barcoding and stable isotope analysis improve prey identification and provide a more quantitative dietary analysis of adult black bass in Lake Ontario, comparing the importance of Round Goby as prey between these two species. Eighty-four LMB (406mm fork length ±4mm SEM) and two hundred sixty-four SMB (422mm ±2mm) obtained as tournament mortalities had prey identified using DNA-based methods. Round Goby was the most prevalent prey species for both predators. The diet of LMB was three times more diverse than that of SMB, which almost entirely consists of Round Goby. Our results provide further support that recent increases in the size of Lake Ontario bass are a result of Round Goby consumption, and that the effects of this dietary shift on body condition are greater for SMB. Techniques developed in this study include: reverse-oriented dual priming oligonucleotides used as blocking primers for predator DNA, and an automated design approach of restriction fragment length polymorphism tests for identifying prey DNA barcodes.