The development of grass cultivars derived from Festulolium hybrids is mostly limited because of their genetic instability and low fertility. The objective of this was to assess the genomic structure and fertility in two advanced breeding populations derived from an interspecific hybrid between Festuca pratensis Huds. (2n = 4× = 28) and Lolium perenne L. (2n = 4× = 28). Examination based on genomic in situ hybridization analyses of randomly chosen plants in the F7 and F8 generations, as well as those from earlier generations, indicated progressive changes in genome balance towards that of Lolium. The ‘dominance’ of Lolium chromatin over Festuca likely resulted from extensive recombination between chromosomes of the parental genomes, together with substitutions of whole Festuca chromosomes by whole Lolium chromosomes. The number of recombinant chromosomes and recombination breakpoints per genotype increased in successive generations, but their number was higher for Festuca than for Lolium. Because the mean seed set was only 38.2% in the F8 generation, it was concluded that the F. pratensis × L. perenne amphiploids cannot be included successfully in a Festulolium breeding programme.