•The MS ortho effect was used for the first time in the analysis of PCBs in fish.•Other new observations on mass spectra have solved old problems often reported.•An accurate gas chromatographic setup was combined with these findings.•The seven indicators were analyzed via GC–MS in a simple but innovative way. A simple instrumental procedure was developed to carry out the not simple analysis of PCBs in fish samples. PCBs with the same degree of chlorination (the isomers) are expected to be totally indistinguishable among them by all existing detectors and by all existing mass spectrometers, and there is no apparent solution in those frequent cases where two isomers chromatographically coelute. Generally such coelutions are solved by means of multidimensional GC, but it is a complex technique impractical for most laboratories. The present research focuses on the seven important “indicator PCBs” by using the mass spectrometer in an innovative way. The “mass spectrometric ortho effect” was usefully exploited in addressing coelutions between isomers. Other new important observations on mass spectra were decisive in solving the apparent problem arising from coelutions between higher chlorinated PCBs with the lower chlorinated ones when low-resolution MS is used. With the proposed procedure, the seven indicators are analyzed in a simple way and with a degree of accuracy never observed with the conventional gas chromatography. The method was applied to some Bluefin Tuna fish samples of big size suspected to have not negligible levels of PCBs due to the high position of this species in the food chain. The supposition was partly confirmed. On the basis of the results here obtained, the recently introduced EU Regulation on six of the seven indicators shows one critical point: in the present paper, an amendment to the Regulation is proposed. A number of important validation measures are reported.