Brassica carinata (BBCC, 2n ¼ 34) possesses many good agronomic characters and, to provide more genetic information about this species and utilize it further, 110 accessions of B. carinata were tested for genetic variation by using 233 amplified fragment length polymorphism markers, so that a dendrogram could be constructed. To combine the good traits of B. carinata and B. rapa (AA, 2n ¼ 20) with those of B. napus (AACC, 2n ¼ 38) which is the major rapeseed variety in China, interspecific crosses between these two species have been made which have resulted in 276 doubled hybrids (AABBCC). These hexaploids combine positive characters and can be used for crossing with B. napus. Brassica carinata as the female parents exhibited extensive differences in interspecific crossability and 20 high crossability accessions were identified. The Poisson Regression Model analysis result (P < 0.0255) indicated that such differences were due to the genotype of the accessions. Accessions with high crossability could promote gene flow within the genus Brassica.