Pyrimidine (Pym, 1,3-diazine, 1,3-diazabenzene) is an important N-heterocyclic building block of nucleobases. Understanding the structures of its fragment and precursor ions provides insight into its prebiotic and abiotic synthetic route. The long-standing controversial debate about the structures of the primary fragment ions of the Pym⁺ cation (C₄H₄N₂⁺, m/z 80) resulting from loss of HCN, C₃H₃N⁺ (m/z 53), is closed herein with the aid of a combined approach utilizing infrared photodissociation (IRPD) spectroscopy in the CH and NH stretch ranges (ν_CH/NH) and density functional theory (DFT) calculations. IRPD spectra of cold Ar/N₂-tagged fragment ions reveal that the C₃H₃N⁺ population is dominated by cis-/trans-HCCHNCH⁺ ions (∼90%) along with a minor contribution of the most stable H₂CCCNH⁺ and cis-/trans-HCCHCNH⁺ isomers (∼10%). We also spectroscopically confirm that the secondary fragment resulting from further loss of HCN, C₂H₂⁺ (m/z 26), is the acetylene cation (HCCH⁺). The spectroscopic characterization of the identified C₃H₃N⁺ isomers and their hydrogen-bonded dimers with Ar and N₂ provides insight into the acidity of their CH and NH groups. Finally, the vibrational properties of Pym⁺ in the 3 μm range are probed by IRPD of Pym⁺-(N₂)_1–2 clusters, which shows a high π-binding affinity of Pym⁺ toward a nonpolar hydrophobic ligand. Its ν_CH spectrum confirms the different acidity of the three nonequivalent CH groups.