The improvement of polymers flames resistance for over fifty years using polybrominated diphenyl ethers (PBDEs) resulted in the environment and food of animal origin pollution. The number of PBDE congeners, their low concentration in food, and potentially co-eluting isobaric compounds require careful selection of chromatographic separation parameters. The aim of this study was the separation and determination of ten individual PBDE congeners in the fish muscle, oil, eggs, and milk required by European law (2014/118/EU). The analytical procedure of ten PBDE congeners quantification was based on the isotope (13C12) dilution method and the gas chromatography with high-resolution mass spectrometry detection. The analytes were extracted, then clean up using self-packed columns. The congeners separation using six capillary gas chromatography columns was checked. Two columns were selected for further study. The first one allowed for the separation of BDE-28 and BDE-154 from disturbing isobaric compounds, while the second one gave the appropriate sensitivity for highly brominated congeners. The method was successfully validated and positively evaluated in international proficiency tests. This work shows how critical is proper chromatographic separation for PBDE determination in food samples. Despite the use of sophisticated analytical techniques, false-positive results can still be obtained.