Technical Abstract: The F1 hybrid offspring of channel catfish, Ictalurus punctatus, females mated with blue catfish, I. furcatus, males contain many desirable traits for commercial production such as enhanced growth and increased survivability. Although at a low efficiency, hybrids can be produced by pond spawning, but hybrid catfish cannot always be readily distinguished from channel catfish, especially at early life stages. The present research was designed to produce a rapid DNA-based test for the identification of hybrid catfish. Channel and blue catfish genomic regions were PCR-amplified using common primers for the follistatin (Fst) and hepcidin antimicrobial protein (Hamp) genes, and fragment length polymorphisms between the two species caused by ancestral insertions/deletions were resolved by agarose electrophoresis. The Fst amplicons were 399 and 348 bp while the Hamp amplicons were 262 and 222 bp from blue and channel catfish, respectively. The mitochondrial cytochrome c oxidase I gene (Mtco1) was also differentially amplified from each species using species-specific primers, thus the maternal parent species could be determined. The DNA preparation technique provided sufficient genomic DNA to test all life stages. The PCR products were successfully amplified from genomic DNA isolated from embryos at one, two, or five days after fertilization, from fry two days after hatching, from blood and barbels of juveniles and adults, and from fresh, frozen, and cooked fillet samples. The results from this assay were available as soon as 24 h after receipt of sample. This assay will be useful for management of hybrid populations and post-harvest detection of hybrid catfish products.