An atypical distribution of steroids occurred in oilsand and oil samples in the Jinxian Sag, Bohai Bay Basin, northern China. It is featured by usually abundant C-21-C-26 short-chain steranes that are derived from the cleavage of the weak C-S bonds at the C-17 and C-20 positions. Sulfur-containing petroleum biomarkers were further analyzed in these sulfur-rich heavy oils by comprehensive two-dimensional gas chromatography (GC X GC) using sulfur-specific detection to locate and confirm sulfur species and GC X GC time-of-flight mass spectrometry (TOF MS) to identify previously unknown sulfur-containing biomarkers. Preliminary GC-MS analysis revealed a series of compounds yielding characteristic fragment ions consistent with thiophenic substructures. For confirmation, GC X GC-sulfur chemiluminescence detection (SCD) was used to confirm the sulfur moiety. The molecular structures of sulfur biomarkers were proposed as unsaturated isoprenoid thiophenes based on fragmentation patterns observed in their 70 eV electron-impact ionization (El) mass spectra. There were constant neutral losses of 71 and 111 Da from the molecular ions in the 70 eV El mass spectra throughout a whole series. The endeavor of sulfur-containing biomarker characterization may provide some clues of paleotransformation of biological precursors and geological products in oil/kerogen under thermal stress during burial and resolve some missing links between the biosphere and the geosphere.