Interspecific hybridization is a powerful approach to broaden genetic diversity and generate novel variations in crops. Apetalous Brassica juncea (AABB, 2n=36) has great interest in breeding programs, but apetalous germplasm is quite rare. In this study, apetalous B. juncea (ABJ) that can stably inherit was obtained by interspecific hybridization between B. juncea and B. carinata (BBCC, 2n=34). Cytological observation showed that the chromosome behaviour of ABJ was normal, with 18 bivalents at diakinesis and chromosome segregation of 18:18 at anaphase І. Moreover, fluorescence in situ hybridization (FISH) using a B genome-specific repeat sequence as a probe showed that the chromosome configuration of ABJ, which comprised 16 B-genome chromosomes and 20 A-genome chromosomes, was consistent with that of the parental B. juncea. However, the combination analysis of RNA-seq and molecular markers eventually demonstrated that chromosome A3 lost a 15.8 Mb fragment in ABJ. Based on the gene expression profile in ABJ, several genes related to petal growth and development were screened out. The apetalous B. juncea was the result of a deleted chromosome fragment was first obtained through interspecific hybridization, which not only provides a novel resource for genetic improvement of apetalous B. juncea but also offers an ideal material for analysing the molecular mechanism of petal development in B. juncea.