Trimethoprim crystallizes in the triclinic space group P-1 (#2) with a = 10.5085(3), b = 10.5417(2), c = 8.05869(13) Å, α = 101.23371(21), β = 112.1787(3), γ = 112.6321(4)°, V = 743.729 Å3, and Z = 2. A reduced cell search in the Cambridge Structural Database yielded three previous structure determinations, using data collected at 100 K, 173 K, and room temperature. In this work, the sample was ordered from the United States Pharmacopeial Convention (USP) and analyzed as-received. The room temperature (295 K) crystal structure was refined using synchrotron (λ = 0.412826 Å) powder diffraction data and optimized using density functional theory techniques. We found similar hydrogen bonding patterns with the previous determinations. In addition, we identified two C–H⋯O hydrogen bonds, which also contribute to the crystal energy. When comparing the previously reported trimethoprim structure determinations, the unit cell length lattice parameters were found to contract at lower temperatures, particularly 100 K. All structures show reasonable agreement, with unit cell length differences ranging between 0.05 and 0.15 Å. The diffraction data for this study were collected on beamline 11-BM at the Advanced Photon Source, and the powder X-ray diffraction pattern of the compound has been submitted to ICDD® for inclusion in the Powder Diffraction File™ (PDF®).