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The Brassicaceae family, commonly referred to as cruciferous plants, is globally cultivated and consumed, with the <i>Brassica</i> genus being particularly renowned for its functional components. These vegetables are rich sources of nutrients and health-promoting phytochemicals, garnering increased attention in recent years. This study presents a comprehensive microscopic, chromatographic, and spectroscopic characterization of <i>Brassica napus</i> L. seeds from Kazakhstan aimed at elucidating their morphological features and chemical composition. Microscopic analysis revealed distinct localization of flavonoids, total lipids, and alkaloids. High-performance thin-layer chromatography (HPTLC) analysis of seed extracts demonstrated a complex chemical profile with significant quantities of non-polar compounds in the hexane extracts. Additionally, methanolic extracts revealed the presence of diverse chemical compounds, including alkaloids, flavonoids, and glucosinolates. The chemical composition exhibited varietal differences across different <i>Brassica</i> species, with <i>B. napus</i> L. seeds showing higher concentrations of bioactive compounds. Furthermore, liquid chromatography–quadrupole time-of-flight mass spectrometry (LC-QToF-MS) analysis provided insights into the chemical composition, with sinapine isomers, feruloyl, and sinapoyl choline derivatives as major compounds in the seeds. This study contributes to a better understanding of the chemical diversity and quality control methods’ approximations of <i>B. napus</i> L. seeds, highlighting their importance in functional food and nutraceutical applications.