Plants were regenerated from a cell suspension from shoot-tip derived callus of a pentaploid Lolium multiflorum (2n = 4x = 28) × Festuca arundinacea (2n = 6x = 42) in which a set of five homologous and homoeologous chromosomes were marked at the PGI/2 locus by distinct alleles. A direct relationship was found in regenerated plants between time in cell suspension and the number of aberrations at the PGI/2 locus. These included deletion of any one, and in a few cases two, of the five PGI/2 alleles, but these were not always related to chromosome loss. In addition three different PGI/2 alleles were each rendered null in some somaclones regenerated last from the cell suspension. While bivalent and trivalent frequency remained unaltered in the regenerated plants compared with the original hybrid, univalent frequency decreased. Chromosome configurations of four or more chromosomes, which probably represent intergeneric chromosome pairing, were significantly increased in the regenerated plants compared with the original hybrid and were negatively correlated with univalents. The possible incorporation of a cell culture phase as a way of increasing intergeneric recombination between L. multiflorum and F. arundinacea chromosomes in a conventional breeding program is discussed.Key words: Festuca–Lolium, somaclonal variation, phosphoglucoisomerase (PGI/2), chromosome instability, somatic recombination.