<p>To investigate the influence of thermal effects on the formation and distribution of sterane compounds in crude oil, we conducted a systematic geochemical analysis of 26 typical marine crude oil samples and one pyrolytic simulation sample from the Tarim Basin. The results reveal significant variations in sterane composition and distribution across maturity stages (Ro: 1.0%–2.5%). During the highly mature stage, thermal cracking significantly reduces sterane content. In the over-mature stage, a transient increase in sterane abundance correlates with the cracking of high-molecular-weight hydrocarbons. As maturity increases, differential generation and cracking rates of C₂₇ and C₂₉ regular steranes drive a characteristic trend: the C₂₇/C₂₉ regular sterane ratio initially decreases then increases. Consequently, this ratio cannot serve as a reliable source indicator in mature to highly mature stages. Similarly, divergent thermal stabilities cause a “reversal” in the C₂₉ sterane isomerization parameter at high maturity. Hence, at high thermal maturity levels, steranes become less applicable due to the equilibrium. Detected sterane isomerization parameters cannot be directly applied to constrain other geochemical effects. Furthermore, the ratio of rearranged to regular steranes exhibits a strong positive correlation with maturity, establishing its utility as an effective maturity parameter for crude oils in mature to highly mature stages.</p>