Brassica carinata originating from Africa with good adaptation in drought and hot environment is an important genetic source to improve B. juncea, which has been widely sown in the Northwest of China. The technique of genomic in situ hybridization (GISH) was adopted to investigate the chromosome segregation of interspecific hybrid (ACBB, 2n = 35) between B. juncea and B. carinata during meiosis. The predominant segregation pattern of chromosomes was 17:18 in pollen mother cells (PMCs), followed by 16:19, while lagged chromosomes were occasionally observed. And the major segregation pattern of B-genome chromosomes from two species was 8:8. Our data suggest that it will be possible to transfer the genetic component of B. carinata into B. juncea via homologous recombination within B genome and homeologous recombination among three genomes.