AbstractSeveral representative pyrimidine derivatives were selected to undergo electrospray ionization (ESI) followed by collision‐induced dissociation tandem mass spectrometry (CID MS/MS) experiments. Two competitive pathways were found to govern the formation of major fragment ions from protonated species of these molecules. The pathways were largely affected by the 2‐O‐methyl group but not significantly influenced by the substitution on C‐5 site of the pyrimidine ring. These findings were supported by both deuterium labeling CID MS/MS experiments and theoretical calculations. The deuterium labeled pyrimidine ion molecules were generated in‐source in ESI from the fully deuterated hydrazinyl pyrimidines, which were readily obtained through hydrogen/deuterium (H/D) exchange when dissolved in deuterium oxide (D2O).