Supernumerary (B) chromosomes of rye are not required for plant development and exhibit a reduced transcription activity. These special features inspired us to analyse whether there are differences between A and B chromatin organization in interphase nuclei. Applying fluorescence in situ hybridization, we found that both rye A and B chromosomes added to hexaploid wheat showed in meristematic nuclei a string-like shape and a clear Rabl orientation. In 4C differentiated leaf nuclei, a more relaxed chromatin structure, round-shaped chromosome territories and a less pronounced Rabl configuration were found. Also, the observed random association of homologues in 2C and 4C nuclei indicated in general a similar behaviour of A and B chromosomes. Whereas in differentiated 4C nuclei A sister centromeres are separated, B sister centromeres align in nearly all nuclei. In short, despite the different transcription activity of A and B chromosomes, both types of chromosomes exhibit a similar organization in meristematic and differentiated interphase nuclei. But the deletion of a B chromosome segment responsible for non-disjunction during gametogenesis induces released sister centromeres also in some interphase nuclei of somatic tissue. Hence, the control of rye B chromosome non-disjunction is also active in sporophytic cells.