A stepwise UPGMA cluster sampling approach with three different genetic similarity coefficients(Jaccard, SM, and Nei & Li) was applied to a SSR molecular data of 92-genotype primary core collection. Results showed that 92 genotypes possessed abundant genetic diversity at 15 SSR loci, which produced 331 polymorphic bands in amplification, constituting a mean of 100% of total bands. The genetic similarity coefficients were constructed with three approaches and two random samplings, which produced five putative core collections. To evaluate the quality of putative core collections, three indices, Shannon-Wiener diversity index, polymorphic band number and percentage of polymorphic bands, using core collections had the highest identification efficiency, while others were less efficient. The putative core collection constructed using SM genetic similarity coefficient had higher quality than others. The putative core collection consisted of 80 Saccharum spontaneum genotypes shaving a 0.984 1 Nei’s diversity index and 4.259 8 Shannon-Wiener diversity index, and did not differ significantly(P<0.05) in molecular diversity from the primary core. Further, MD was 0%(<20.00%) and CR 99.16%(>80.00%).The putative core collection of 80 S. spontaneum genotypes based on SM genetic similarity coefficient did not differ significantly from the genetic diversity of the primary core regarding to the agronomic traits or molecular markers. The putative core will facilitate the evaluation and utilization of the germplasm in developing elite sugarcane hybrids, and mining elite genes.