The quantification of species in commercial products is limited by analytical shortcomings, as most of them provide semiquantitative results. An exception is real-time PCR, which can provide quantitative results using hybridization probes. In the present work, this technique has been applied to the absolute, absolute-relative and relative quantification of the most valued hake species in European markets, Merluccius merluccius (European Hake). The best quantification results for this species in binary mixtures with non-target species (Merluccius capensis) and using a species-specific real-time PCR MMER_VIC system was achieved using a relative quantification approach (MLL as reference system). Absolute quantification using the MLL nuclear system has been demonstrated as appropriate for the quantification of the Merluccius genus in food model samples. This study reveals the impact of different reference systems (MLL and HAKE) in the absolute-relative and relative quantification approaches, showing that the nuclear MLL system performed better than the mitochondrial HAKE system.