Molecular characterization of sulfur-containing species in petroleum is important because sulfur-containing compounds are detrimental to the environment and the refining processes. In a recent report, the sulfur-containing compounds in a vacuum bottom residue (VBR) were methylated to enhance their delectability by electrospray ionization (ESI) mass analysis. The most abundant sulfur compounds exhibited relatively low double bond equivalents (4 < DBE < 12). Alternatively, atmospheric pressure photoionization (APPI) mass analysis can provide molecular characterization without chemical derivatization. Here, we compare the sulfur speciation of a petroleum vacuum bottom residue by ESI and APPI with a 9.4 T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Even after methylation, ions produced by APPI extend to much higher DBE than by ESI. Moreover, analysis of the saturates and aromatics fractions of underivatized VBR by APPI shows comparable ionization efficiency across a broad DBE range. We conclude that methylation is hindered for high-DBE species (DBE > 20), so that methylation followed by ESI MS is not suitable for sulfur speciation of higher-boiling fractions from petroleum crude oil.