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Thirty-nine crude oils and twenty-one rock samples from Niger Delta Basin, Nigeria have been characterized based on their isotope compositions by elemental analysis-isotope ratio mass spectrometry and gas chromatography-isotope ratio mass spectrometry. The bulk carbon isotopic values of the whole rock extracts, saturate and aromatic fractions range from –28.7‰to –26.8‰, –29.2‰to –27.2 ‰and–28.5‰ to –26.7‰, respectively while the bulk carbon isotopic values of the whole oils, saturate and aromatic fractions range from –25.4‰ to –27.8‰, –25.9‰ to –28.4‰ and –23.5‰ to –26.9‰, respectively. The average carbon isotopic compositions of individual alkanes (nC12-nC33) in the rock samples range from–34.9‰to–28.2‰whereas the average isotopic values of individual n-alkanes in the oils range from –31.1‰to –23.8‰. Theδ13C isotope ratios of pristane and phytane in the rock samples range from –29.2‰ to –28.2‰ and –30.2‰ to –27.4‰ respectively while the pristane and phytane isotopic values range from–32.1‰to–21.9‰and–30.5‰to–26.9‰, respectively. The isotopic values recorded for the samples indicated that the crude oils were formed from the mixed input of terrigenous and marine organic matter and deposited under oxic to sub-oxic condition in lacustrine-fluvial/deltaic environments. The stable carbon isotopic compositions were found to be effective in assessing the origin and depositional environments of crude oils in the Niger Delta Basin.