A method for the unambiguous determination of 41 key polychlorinated biphenyls (PCBs) (including coplanar and chiral congeners) and the enantiomeric ratio of chiral congeners is described. The method includes a fractionation step using a 2-(1-pyrenyl) ethyldimethylsilylated silica column for separating the polychlorinated biphenyls according to the number of chlorine atoms in the ortho positions. High-resolution gas chromatography with an electron capture detector and an achiral column was used to determine the PCB congener content in each fraction. The enantiomeric ratio of chiral congeners was calculated by high-resolution gas chromatography with a mass spectrometry detector using a chiral column. The method was found to be inexpensive, rapid, effective, and reliable under the operational conditions proposed. It eliminates the main coelution problems among the polychlorinated biphenyl congeners. It also makes it possible to determine the enantiomeric ratio of nine chiral congeners using monodimensional gas chromatography. The method was applied successfully to the analysis of the coplanar and atropisomeric polychlorinated biphenyl congeners in dolphin liver samples. The enantiomeric ratio of nine chiral congeners is also reported for the first time.