Abstract: Trimethyl-alkylbenzenes are important molecular markers and are of great significance for interpreting depositional environments and thermal maturity. However, their identification and application are challenging due to co-elution problems caused by structural similarities, as well as the effects of biodegradation and thermal alteration. This study focused on oils and asphaltenes with varying degrees of biodegradation from the Tarim Basin. By comparing asphaltene-bound hydrocarbons with free hydrocarbons, this study systematically investigated the differential distribution characteristics and genetic implications of multiple series of trimethyl-alkylbenzenes, including aryl isoprenoids. To address the co-elution problem caused by structural similarities, chromatographic columns with different polarities could effectively improve separation. Furthermore, combining the mass spectrometric characteristics of multi-series trimethyl-alkylbenzenes enabled more accurate identification of various isomers. The 2,3,6-trimethyl-aryl isoprenoid (2,3,6-TMIPB), 2,3,6-trimethyl-isohexylbenzene (2,3,6-TMiHB), and trimethyl-n-alkylbenzene (TMnAB) series were widely detected in asphaltene-bound hydrocarbons of biodegraded oils/asphaltenes from the Tarim Basin. Among them, 2,3,6-TMIPB and 2,3,6-TMiHB were widely present in oils and asphaltenes from both the interior and margin of the basin, indicating that the organic matter of source rocks had similar depositional environments and parent material sources during source rock deposition. Additionally, it demonstrated that asphaltene-bound hydrocarbons were key to investigating the origin of biodegraded oils. Significant differences in the relative abundances of trimethyl-alkylbenzenes between free and asphaltene-bound hydrocarbons were observed in oil and asphaltene samples from Tarim Basin, reflecting variations in the degree of biodegradation, the thermal maturity of oils from different charging episodes, and the extent of secondary cracking of early-charged oils. In ancient petroliferous basins, the superimposition of multiple source rock systems and multi-stage hydrocarbon accumulation events led to the widespread occurrence of post-biodegradation oil re-charging and mixing. This study provides new evidence for reconstructing the oil charging and mixing history in biodegraded reservoirs, and has positive implications for assessing multi-phase oil alteration and evaluating resource exploration potential.