Festulolium is an intergeneric hybrid between Festuca and Lolium spp. that has agronomic benefits derived from both grass species. The genomic constitution of this intergeneric hybrid has been examined previously using genomic in situ hybridization (GISH). However, the genomic constitution of the first festulolium cultivar developed in Japan, ‘Tohoku 1’, which was released in 2009, has not yet been determined by GISH. We therefore developed a method for determining the genomic constitution of festulolium hybrids using multi-colored GISH and image analysis. GISH revealed numerous recombinant chromosomes with a relatively small proportion of the Festuca genome in Tohoku 1. Also, discriminating the origin of the species based on staining of the centromeres and nucleolar organizing regions in festulolium by GISH and FISH with an rDNA probe is difficult. The ratio of the Festuca-specific genome (f ratio) was tested for use as an evaluation index for estimating the genomic constitution of festulolium. Although the observed number of Festuca chromatins in Tohoku 1 was reduced during mitosis, the f ratio was stable throughout mitosis, indicating that the f ratio is a useful index for characterizing the festulolium genome.