Glucosinolates (GSL) are important secondary metabolites in Brassicaceae, with favorable biological functions such as resistance to plant diseases and insect pests. In this paper, we investigated and compared the content of 11 major GSL among three major organs (roots, leaves and seeds) of 67 cultivars using high-performance liquid chromatography (HPLC). These cultivars were from four Brassica oilseed species (B. napus L., B. carinata L., B. juncea L. and B. rapa L.) and cultivated at three environments (Wuhan Hubei, 2014; Wuhan Hubei, 2016; Xiangyang Hubei, 2016). In general, the difference between years resulted in extremely significant variation of GSL content. Cultivars from different species showed significant difference on type of GSL. In comparison to the cultivars from B. napus L. and B. carinata L. that showed extremely significant variation of GSL content, no significant variations were observed in cultivars from B. juncea L.and B. rapa L.. For the same cultivar, the total content of GSL among different organs varied significantly and for the same organ, the content of different GSL varied significantly as well. These results showed that there is abundant variation of GSL content or types among different species, organs, and cultivars as well as environmental factors. There are a few special individual GSL in certain species and organs, and some of them showed beneficial biological functions, which would be transferred and accumulated in B. napus L. via hybridization between species. For instance, sinigrin, which confers resistance to microorganisms, was typically detected in B. carinata L. and B. juncea L., but almost undetectable in normal B. napus L. cultivars which can be divided into low-sulfur, middle-sulfur and high-sulfur types. These compounds were found in the resynthesized lines of B. napus L.with subgenomic introgression from B. carinata L. and B. rapa L.. Out of 183 lines including three generations, 170 showed an elevation in sinigrin content relative to normal cultivars. Taken together, these results would provide new insights for deploying GSL in different oilseeds crops in future breeding for new Brassica crops.