In vitro variation of B chromosomes was studied by examining the callus cells derived from the immature embryos from a cross of Chinese Spring wheat (Triticum aestivum L.) and Fin 7416 rye (Secale cereale L.) carrying two B chromosomes. In 40-d-old callus cells, the numbers of B chromosomes ranged from one to four in 65.6% of the cells observed. The distribution of B chromosome numbers was associated with the ploidy levels of the normal chromosomes (A chromosomes). The frequency of the cells with high numbers of B chromosomes (i.e., three or four B chromosomes) in the amphiploid cells with 56 A chromosomes was greater than those in the haploid cells with 28 A chromosomes. Although structural changes in the rye A chromosomes were observed, cytological observation and genomic in situ hybridization demonstrated that the rye B chromosomes were conserved in morphological appearance following tissue culture.