The 45S ribosomal DNA (rDNA) units are separated by an intergenic spacer (IGS) containing the signals for transcription and processing of rRNAs. For the first time, we sequenced and analyzed the entire IGS region from three original species within the genus Saccharum, including S. spontaneum, S. robustum, and S. officinarum in this study. We have compared the IGS organization within three original species of the genus Saccharum. The IGS of these three original species showed similar overall organizations comprised of putative functional elements needed for rRNA gene activity as well as a non-transcribed spacer (NTS), a promoter region, and an external transcribed spacer (ETS). The variability in length of the IGS sequences was assessed at the individual, intraspecies, and interspecies levels of the genus Saccharum, including S. spontaneum, S. robustum, and S. officinarum. The ETS had greater similarity than the NTS across species, but nevertheless exhibited variation in length. Within the IGS of the Saccharum species, base substitutions and copy number variation of sub-repeat were causes of the divergence in IGS sequences. We also identified a significant number of methylation sites. Furthermore, fluorescent in situ hybridization (FISH) co-localization of IGS and pTa71 probes was detected on all representative species of the genus Saccharum tested. Taken together, the results of this study provide a better insight into the structure and organization of the IGS in the genus Saccharum.