Sugarcane (Saccharum spp.) is probably the crop with the most complex genome. Modern cultivars (2n=100-120) are derived from interspecific hybridization between the noble cane S. officinarum (2n=80) and the wild cane S. spontaneum (2n=40-128). We investigated the genome organization of important sugarcane cultivars and their parental species using chromosomespecific probes combined with genomic in situ hybridization (GISH). This allowed the genomic and genetic characterisation of Australian sugarcane cultivars and one of the major contributing parental clones, Mandalay. The S. spontaneum clone Mandalay follows the classical organization of S. spontaneum clones with x=8 with a major discrepancy related to an extra six chromosomes compared to the previously reported 2n=96 for Mandalay’s clone. Our previous results reported the rearrangements between the S. officinarum (x=10) and S. spontaneum (x=8) chromosomes, with a most likely scenario of a two-step process leading to x= 9 and then x=8, where each step involved three chromosomes that were rearranged into two. Further polyploidization led to the wide geographical dispersion of S. spontaneum clones with x= 8. In modern cultivars, the 13-20% of the S. spontaneum contribution originated from cytotypes with x=8. Modern cultivars have mainly 12 copies of each of the first four basic chromosomes and a more variable number for those basic chromosomes whose structure differs between the two parental species. These new insights and cytogenetic tools substantially improve our understanding of the extreme level of complexity of modern sugarcane cultivar genomes and could lead to guiding breeding strategies in the development of new improved varieties for the Australian industry.