Diagnostic molecular markers are an essential tool in the study of species’ ecology and evolution, particularly in closely related and sympatric species. Furthermore, the increasing awareness of wild-hybrids has led to a renewed interest in rapid diagnostic assays. Here, we test the ability of two mitochondrial (Cytb and COI) and two nuclear markers (ETS2 and TMO-4c4) to confidently discriminate purebred P. leopardus and P. maculatus and their first-generation hybrids. A sample of 48 purebred individuals and 91 interspecific hybrids were used in this study and their delineation confirmed using a set of microsatellite markers. Our results indicate mitochondrial markers could not distinguish even between species but both nuclear markers confidently identified species and first-generation hybrids. However, later-generation hybrids could not always be confidently identified due to on-going introgression between species. Our findings provide a robust tool to distinguish purebred individuals and interspecific hybrids in a pair of species with an unexpectedly high incidence of hybridization. The quick species discrimination abilities provided by these diagnostic markers are important for stock assessment and recruitment studies of these important fishery species.