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This present paper includes a detailed evaluation of specific biomarkers together with stable carbon isotope (δ13C) by gas chromatography-mass spectrometry (GC–MS) and Gas Chromatograph–Isotope Ratio Mass Spectrometry (GC–IR–MS). Eight crude oil samples were collected from the A, B, H (east) and H (west) Fields, located in the Murzuq Basin, Libya. Stable Carbon isotope data (δ13C) together with biomarker ratios data of individual hydrocarbons, n-alkanes, isoprenoids, terpenes, hopanes, steranes and aromatic have been determined in crude oils to delineate their bacterial degradation, source facies, organic matter precursors, depositional conditions and a variation of maturation. Based on source-specific parameters including n-C19 alkane, % C27S, %C28S, %C29S, %C23TT, %C30αβ, %rC28, DBT/P, CPI, Pr/Ph, Ts/Tm, dh 30/h 30, 1 MN, 2 MN, 26-27 DMN, 15 DMN, 236 TMN, 146-135 TMN, 125 TMN, 136 TMN ratios and δ13C‰ of saturates and aromatics fractions. Such oils showed non-biodegradation, n-C19 peak proved oils generated from a Lower Palaeozoic source rocks as emphasizedvian-C19 peak, the dominance of C29steranes over C27 and C28 with light Carbon isotope ratio (δ13C‰) values. The abundances of isosterane C29, C27, C28.Tricyclohexaprenol and bacteriohopane polyols and aminopolyols, recommended as mixture bioprecursors of tricyclic terpenes and hopanes, furthermore regular sterane ratio gives values characteristic of Lower Palaeozoic marine source rocks and holding green algae and most likely a quantity of contribution from acritarchs. Carbon preference indices (CPIs)>0.9 pointed to an anoxic deposition, dibenzothiophene to phenanthrene (DBT/Prange 0.49 - 0.58) recommend a siliciclastic source rather than carbonate and/or evaporate saline deposition. The ratios of CPIs, pristane/n-C17 and phytane/n-C18, n-alkanes (C16 to C22) against (C23 to C33), Ts/Tm, C30diahopane/C30hopane, methylnaphthalene, dimethyl naphthalene and trimethyl naphthalene indicated that the oils analysed are mature except the B Field oil being slightly less mature than the A, H (east) and H (west) Fields oils.