The reported source rocks for the abundant petroleum in the Tarim Basin, China range from Cambrian to Lower Ordovician and/or Upper Ordovician in age. However, the difference between the two groups of source rocks is not well characterized. In this study, pyrite was removed from eleven mature to over mature kerogen samples from source rocks using the method of CrCl2 reduction and grinding. The kerogen and coexisting pyrite samples were then analyzed for δ34S values. Results show that the kerogen samples from the Cambrian have δ34S values between +10.4‰ and +19.4‰. The values are significantly higher than those from the Lower Ordovician kerogen (δ34S of between +6.7‰ and +8.7‰), which in turn are generally higher than from the Upper Ordovician kerogen samples (δ34S of between -15.3 and +6.8‰). The associated pyrite shows a similar trend but with much lower δ34S values. This stratigraphically controlled sulfur isotope variation parallels the evolving contemporary marine sulfate and dated oil δ34S values from other basins, suggesting that seawater sulfate and source rock age have an important influence on kerogen and pyrite δ34S values. The relatively high δ34S values in the Cambrian to Lower Ordovician source rocks are associated with abundant aryl isoprenoids, gammacerane and C35 homohopanes in the extractable organic matter, indicating that these source rocks were deposited in a bottom water euxinic environment with water stratification. Compared with the Upper Ordovician, the Cambrian to Lower Ordovician source rocks show abundance in C28 20R sterane, C23 tricyclic terpanes, 4,23,24-trimethyl triaromatic dinosteroids and depletion in C24 tetracyclic terpane, C29 hopane. Thus, δ34S values and biomarkers of source rock organic matter can be used for distinguishing the Cambrian and Upper Ordovician source rocks in the Tarim Basin.