Sugarcane from diverse ecological regions exhibits significant genetic diversity and is considered a rich source of novel or unique alleles. Microsatellites, also referred to as simple sequence repeats (SSRs), are DNA markers characterized by brief, repetitive motifs with varying repeat numbers at specific loci. These highly reproducible markers are crucial tools for genetic analysis. The genetic diversity of germplasm resources plays a vital role in crop improvement programs, as it determines the potential for sustained genetic advancements. SSR fingerprinting is widely employed in genetic diversity studies due to its high level of polymorphism. It allows for identifying allelic variations even among closely related varieties, enabling accurate assessment of genetic diversity in different sugarcane varieties. SSR analysis provides valuable insights into the unique genetic patterns and relationships between different varieties by targeting specific regions of the sugarcane genome. The molecular data generated from the analysis offers crucial guidance in various aspects of sugarcane breeding. It aids in selecting elite parent genotypes for crossing, establishing effective cross-combinations, designing hybrid breeding strategies, and helping address the challenges posed by climate change. Furthermore, the diverse sugarcane panel holds potential for further exploration in genome-wide association studies (GWASs), opening up new avenues for research and crop improvement.