Seafood substitution is a worldwide problem due to factors such as limited monitoring coupled with complex supply chains. Red snapper (Lutjanus campechanus) is a highly valued and overfished species that is commonly substituted with other fish, such as tilapia, rockfish, and other snapper species. DNA barcoding is typically used by regulatory agencies to detect seafood substitution; however, it is expensive and time-consuming. A rapid, real-time PCR assay targeting red snapper was developed previously for use in fisheries management; however, it has not been tested for its ability to detect red snapper species substitution. The objective of this study was to assess the ability of the real-time PCR assay to identify red snapper fillets and differentiate red snapper from common substitute fish species in combination with DNA barcoding. A total of 21 fresh/frozen fillets labeled as “red snapper” were tested with real-time PCR, along with 57 fresh/frozen fillets representing 15 of the most common categories of fish mislabeled as red snapper. All samples were tested with DNA barcoding to confirm the identity of fish species. Real-time PCR parameters were optimized to reduce background signals associated with cross-reactivity. Overall, real-time PCR identified 4 samples as red snapper: 3 were authenticated as red snapper with DNA barcoding and 1 was identified as mahi-mahi. Overall, 40% of all samples and 91% of “red snapper” samples were considered mislabeled according to DNA barcoding. Red snapper was substituted with other snapper species (e.g., Lutjanus malabaricus, Lutjanus peru, Ocyurus chrysurus, and Rhomboplites aurorubens), rockfish (Sebastes flavidus and Sebastes brevispinis), sea bream (Pagrus major/Pagrus auratus), and mahi-mahi (Coryphaena hippurus). The real-time PCR assay tested in this study can serve as a rapid screening test for the detection of mislabeled species, which can then be confirmed with sequencing techniques. This species identification technique has the potential to be used by regulatory agencies to rapidly determine the authenticity of red snapper on-site.