Thermal maturity level of crude oil samples is commonly investigated using popular biomarkers such as steranes and hopanes. Although crude oil's thermal maturity is an important goal for petroleum engineers and geochemists, fewer researches have been published about crude oil maturity than kerogen, source rock, and coal samples. This paper aims to assess the crude oil thermal maturity by asphaltene fraction analyzed by Raman spectroscopy and X-ray diffraction (XRD) methods for the first time. For this goal, the thermal maturity levels of three crude oil samples were investigated by geochemical parameters. In the next step, the asphaltene fractions of studied samples were precipitated and analyzed by two known Raman spectroscopy and X-ray diffraction methods to assess the applications of asphaltene fraction in determining the level of thermal maturity. Raman spectra indicated that during the maturation of organic matters, the amounts of disordered structures of asphaltenes diminish, and the position of the D1 band, as well as its area and intensity, are considered as maturity indicators. The Raman band separation (RBS) between two D1 and G bands, ratios of intensity and area of D1/G, and G's width are influenced by maturity. In this study, the RBS values increase from 210 cm−1 to 237 cm−1 by maturity increment. The results of XRD showed that the average distance between aromatic sheets (dm), the average height of the cluster perpendicular to the plain of sheets (Lc), aromaticity, and the ratio of intensities of two prominent detected bands of 002 and γ (I002/Iγ) are dependent on the maturity variations. Maturity increment resulted in increasing aromaticity (from 23% to 40%), I002/Iγ (from 0.24 to 0.34), and dm as well as decreasing Lc (from 33.84 °A to 29 °A) values. Since there is a good positive correlation between maturity and aromaticity and I002/Iγ, the cross plot of aromaticity versus I002/Iγ can be applied as a novel and proper tool for assessing the maturity of crude oil samples by the XRD patterns of their asphaltene fractions. Eventually, the results of this paper represent that RBS, intensity and area ratios of D1/G bands, G's width, aromaticity, Lc, and I002/Iγ parameters can be implemented for crude oil maturity assessment. The findings of this study can help for a better understanding of crude oil maturity level by the asphaltene fraction through a simple and inexpensive procedure.