Abstract: mbination in local sugarcane cross breeding, SSR-capillary electrophoresis/fluorescence detection (SSR-CE/FD) technology was used to analyze twelve sugarcane genotypes from Guizhou. The SSR data were then calculated using NTsys software. A total of 131 DNA fragments were amplified through PCR by 21 pairs of highly polymorphic SSR primers, of which 90.1% were polymorphic. The number of fragments per primer pair was 6.2. Eleven fragments were genotype-specific fragment of one cultivar (line) from seven cultivars (lines); these genotype-specific fragments could distinguish one cultivar (line) from other six cultivars (lines). The SMC31CUQ primer pair, produced 100% polymorphic alleles, was the highest-resolution primer that could distinguish all the 12 sugarcane genotypes. According to the cluster results of UPGMA program, pairwise similarity coefficients among the 12 molecular identities varied from 0.64 to 0.92. Our study successfully constructed a SSR fingerprint of 131 DNA fragments about 3 cultivars and 9 regional materials in Guizhou Province. The twelve sugarcane genotypes have a narrow genetic base, indicating parental materials with big differences in genetic background should be employed in sugarcane breeding programs in order to broaden the genetic basis among sugarcane cultivars (clones) in Guizhou Province.