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Summary Karyotypes provide key cytogenetic information on the phylogenetic relationships and evolutionary origins in related eukaryotic species. Despite our knowledge of the chromosome numbers of sugarcane and its wild relatives, the chromosome composition and evolution among the species in theSaccharumcomplex have been elusive owing to the complex polyploidy and the large numbers of chromosomes of these species.Oligonucleotide‐based chromosome painting has become a powerful tool of cytogenetic studies especially for plant species with large numbers of chromosomes. We developed oligo‐based chromosome painting probes for all 10 chromosomes inSaccharum officinarum(2n = 8x = 80). The 10 painting probes generated robust fluorescencein situhybridization signals in all plant species within theSaccharumcomplex, including species in the generaSaccharum,Miscanthus,NarengaandErianthus.We conducted comparative chromosome analysis using the same set of probes among species from four different genera within theSaccharumcomplex. Excitingly, we discovered several novel cytotypes and chromosome rearrangements in these species.We discovered that fusion from two different chromosomes is a common type of chromosome rearrangement associated with the species in theSaccharumcomplex. Such fusion events changed the basic chromosome number and resulted in distinct allopolyploids in theSaccharumcomplex.