Abstract Background Karyotype dynamics driven by chromosomal rearrangements has long been considered as a fundamental question in the evolutionary genetics. Saccharum spontaneum , the most primitive and complex species in the genus Saccharum , has reportedly undergone at least two major chromosomal rearrangements, however, its karyotypic evolution remains unclear. Results In this study, four representative accessions, i.e., hypothetical diploid sugarcane ancestor (sorghum, x = 10), Sa. spontaneum Np-X (x = 10, tetraploid), 2012–46 (x = 9, hexaploid) and AP85–441 (x = 8, tetraploid), were selected for karyotype evolution studies. A set of oligonucleotide (oligo)-based barcode probes was developed based on the sorghum genome, which allowed universal identification of all chromosomes from sorghum and Sa. spontaneum . By comparative FISH assays, we reconstructed the karyotype evolutionary history and discovered that although chromosomal rearrangements resulted in greater variation in relative lengths of some chromosomes, all chromosomes maintained a conserved metacentric structure. Additionally, we found that the barcode oligo probe was not applicable for chromosome identification in both Sa. robustum and Sa. officinarum species, suggesting that sorghum is more distantly related to Sa. robustum and Sa. officinarum compared with Sa. spontaneum species. Conclusions Our study demonstrated that the barcode oligo-FISH is an efficient tool for chromosome identification and karyotyping research, and expanded our understanding of the karyotypic and chromosomal evolution in the genus Saccharum .