Yellow-seeded Brassica napus was for the first time developed from interspecific crosses using yellow-seeded B.juncea (AABB), yellow-seeded B.oleracea (CC), and black-seeded artificial B.napus (AACC).Three different mating approaches were undertaken to eliminate B-genome chromosomes after trigenomic hexaploids (AABBCC) were generated.Hybrids (AABCC, ABCC) from crosses AABBCC × AACC, AABBCC × CC and ABCC × AACC were advanced by continuous selfing in approach 1,2 and 3, respectively.To provide more insight into Brassica genome evolution and the cytological basis for B.napus resynthesis in each approach, B -genome chromosome pairing and segregation were intensively analyzed in AABCC and ABCC plants using genomic in situ hybridization methods.The frequencies at which B-genome chromosomes underwent autosyndesis and allosyndesis were generally higher in ABCC than in AABCC plants.The difference was statistically significant for allosyndesis but not autosyndesis.Abnormal distributions of B-genome chromosomes were encountered at anaphase Ⅰ, including chromosome lagging and precocious sister centromere separation of univalents.These abnormalities were observed at a significantly higher frequency in AABCC than in ABCC plants, which resulted in more rapid B -genome chromosome elimination in the AABCC derivatives.Yellow or yellow-brown seeds were obtained in all approaches, although true-breeding yellow-seeded B.napus was developed only in approaches 2 and 3.The efficiency of the B.napus construction approaches was in the order 1 > 3 > 2 whereas this order was 3 > 2 > 1 with respect to the construction of yellow-seeded B.napus.The results are discussed in relation to Brassica genome evolution and the development and utilization of the yellow-seeded B.napus obtained here.