Cultivated sugarcane clones (Saccharum spp., 2n=100 to 130) are derived from complex interspecific hybridizations between the species S. officinarum and S. spontaneum. Using comparative genomic DNA in situ hybridization, we demonstrated that it is possible to distinguish the chromosomes contributed by these two species in an interspecific F1 hybrid and a cultivated clone, R570. In the interspecific F1 studied, we observed n + n transmission of the parental chromosomes instead of the peculiar 2n + n transmission usually described in such crosses. Among the chromosomes of cultivar R570 (2n = 107-115) about 10% were identified as originating from S. spontaneum and about 10% were identified as recombinant chromosomes between the two species S. officinarum and S. spontaneum. This demonstrated for the first time the occurrence of recombination between the chromosomes of these two species. The rDNA sites were located by in situ hybridization in these two species and the cultivar R570. This supported different basic chromosome numbers and chromosome structural differences between the two species and provided a first bridge between physical and genetical mapping in sugarcane.