Available standards provided an opportunity to test all 209 PCB congeners for their mass spectrometric ortho effects, i.e., the percent enhancement of the [M−Cl]+ ion abundances, relative to the molecular ion [M+] abundances for PCB congeners having chlorines in the 2,2′- and 2,2′,6-substituted positions. This work confirmed substantial ortho effects for 84 congeners from di- to nona-chlorinated PCBs. The ortho effect values calculated as ([M−Cl]+/[M+]×100) ranged from 17% to 94%. This study also found that much smaller ortho effects were observed for PCBs with four ortho chlorines (2,2′,6,6′-substitution), for two ortho chlorines on the same ring (2,6-substitution), and for one ortho chlorine (2-substitution), or no ortho chlorines. The ortho effect was also shown to increase for 3- or meta-substitution, increase by a smaller amount for 5-substitution, and decrease considerably for 4- or para-substitution on the biphenyl ring. Mechanistic implications were discussed. Comparison with the NIST 08 and NIST 05 Mass Spectral Libraries showed similar trends, with a few exceptions, that established the value of the ortho effect for proofing library entries. Gas chromatographic studies confirmed previously recognized chromatographic ortho effects on elution and ring substitution/retention order correlations. The order of elution was combined with the measured mass spectrometric ortho effects to test the combination as an additional PCB isomer distinguishing tool for environmental analysis, standards verification, and library proofing.