Genetic diversity analysis, which refers to the elaboration of total extent of genetic characteristics in the genetic makeup of a certain species, constitutes a classical strategy for the study of diversity, population genetic structure, and breeding practices. In this study, fluores-cence-labeled seven gSSR and eight EST-SSR primer pairs and a capillary electrophoresis genotyping platform were used to assess the genetic diversity among 181 sugarcane clones. The clones were sorted into 14 series based on their origin. A total of 205 polymorphic SSR alleles were identified. The mean polymorphic information content (PIC) value was 0.94 for gSSRs and 0.93 for EST-SSRs, respectively. Gene differentiation coefficient (Gst) of inter-series variation (13.71 %) was much lower than intra-series variation (86.29 %). Gene flow value (Nm = 3.15) suggested that there was no significant genetic differentiation or population structure variations among the 14 series. The 181 clones could be clustered into seven groups based on neighbor-joining cluster analysis. Three major groups, namely the USA Group, the Guangxi-Hainan-Fujian Group, and the Guangdong Group, consisted of 36, 64, and 39 clones, respectively. The genotyping data provide valuable information for selecting cross parents, designing cross combinations, and future hybrid breeding strategies.