Determination of the molecular structures of petroporphyrins has been crucial to understand the diagenetic pathways and maturation of petroleum. However, these studies have been hampered by their structural complexity and the challenges associated with their isolation. In comparison to the skeletal macrocyclic structures, much less is known about the substitutions, which are more sensitive to the maturation and diagenesis pathways. While these isolated vanadyl petroporphyrins largely consist of etioporphyrin and deoxophylloerythroetioporphyrin as expected, surprisingly, we find evidence that one or a few β hydrogens are present in petroporphyrins of low carbon numbers using a combination of ultraviolet-visible spectroscopy, Fourier transform ion cyclotron resonance mass spectrometry, and non-contact atomic force microscopy. Petroporphyrins with β hydrogens were not anticipated on the basis of their biological precursors. The data support dealkylation under catagenesis but not transalkylation or random alkylation of the β and meso positions, despite the fact that more complex porphyrin structures are formed.