Kazakhstan ranks as the 12th largest oil producer globally and boasts a diverse range of crude oils. This research introduces a method for distinguishing between the different types of crude oils based on biomarker analysis of 28 crude oils from Western and Southern Kazakhstan using gas chromatography-mass spectrometry. Biomarkers serve as valuable tools, especially in forensic investigations of oil spills. These biomarkers effectively retain a significant portion of the original natural product’s carbon structure, providing crucial evidence regarding the origin and identity of the oils under examination. This study identifies a set of biomarkers, including pristine, phytane, n-C17 and n-C18 alkanes, hopanes, bisnorhopanes, iso-copalanes, pregnane, androstane, allopregnane, homopregnane, cholestane, and stigmastane. By examining ratios such as pristane/phytane, pristane/n-C17 alkane, tricyclic/pentacyclic terpanes, and hopane, as well as the distribution of steranes, it was deduced that crude oils from West Kazakhstan exhibited resilience to biodegradation. These findings showed that gas chromatography-mass spectrometry is an effective method for oil biomarkers determination, especially because it provides efficient separation and identification. Additionally, this study delved into the origin conditions and maturity of these oils, contributing to a deeper understanding of their characteristics and analysis that is simple to use and available worldwide.