Glucosinolates are important secondary metabolites in cruciferous vegetables. In various cruciferous crops, glucosinolates and their degradation products may be different, thus rendering their respective special flavors and biological functions. A main glucosinolates in radish, glucoraphenin(RAE) and its degradation product have powerful anticancer activity, whereas oxazolidinone, a degradation product of progoitrin (PRO), a main glucosinolate in Chinese cabbage, is considered to have the side effect of causing goiters. In this study, high performance liquid chromatography (HPLC) was used to analyze the main components and contents of glucosinolates in radish (‘36-2’), Chinese cabbage (‘Chiifu’) and raphanobrassica, a synthetic intergeneric distant hybrid (DH). A total of 13 glucosinolates were identified. The main glucosinolates in radish were glucoraphasatin (RSA) and RAE. The main glucosinolates in Chinese cabbage were PRO, gluconapin (NAP) and glucobrassicin (GBC). The main glucosinolates in the synthetic intergeneric hybrid species were RAE, glucobrassicanapin (GBN), NAP, PRO and GBC. Through the transcriptome analysis comparing DH with ‘36-2’, it was found that there were 14 down-regulated expression and 23 up-regulated expression genes for glucosinolates pathway in DH, among which the radish-specific RsIQD1.1 and RsMYB34.2 were down-regulated, while RsMYB34.1 was up-regulated. Notably, the RsFMOGS-OX2.1 of radish was significantly up-regulated in DH. Through comparative analysis with ‘Chiifu’, it was found that 24 glucosinolates pathway genes in DH were down-regulated and 15 were up-regulated, among which the Chinese-cabbage-specific BrIPMI-SSU3.1, BrIPMDH3.1 and BrMAM3.1 were significantly up-regulated. The expression characteristics of these genes in DH appeared to be related to the change of the composition of glucosinolates. This proved to some extent that distant hybridization may change the component and content of glucosinolates by changing the genetic composition and gene expression of the offspring, which lays a theoretical foundation and technical support for realization of gene exchanges among species by using distant hybrids as a bridge germplasm and creation of new germplasms of high quality in cruciferous crops.