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Phytosterols are natural compounds found in all higher plants that have a wide variety of roles in plant growth regulation and stress tolerance. The phytosterol composition can also influence the development and reproductive rate of strict herbivorous insects and other important agronomic traits such as temperature and drought tolerance in plants. In this study, we analysed the phytosterol composition in 18 Brassica napus (Rapeseed/canola) cultivars and 20 accessions belonging to 10 related wild Brassicaceae species to explore diverse and novel phytosterol profiles. Plants were grown in a controlled phytotron environment and their phytosterols were analysed using a saponification extraction method followed by GC-MS from the leaf samples. The B. napus cultivars showed slight diversity in eight phytosterols (>0.02%) due to the genotypic effect, whereas the wild accessions showed significant variability in their phytosterol profiles. Of interest, a number of wild accessions were found with high levels of campesterol (HIN20, HIN23, HUN27, HIN30, SARS2, and UPM6563), stigmasterol (UPM6813, UPM6563, ALBA17, and ALBA2), and isofucosterol (SARS12, SAR6, and DMU2). These changes in individual phytosterols, or ratios of phytosterols, can have a significant implication in plant tolerance to abiotic stress and plant insect resistance properties, which can be used in breeding for crop improvement.