Genetic variability among sugarcane genotypes from the Karst region of China was evaluated using genotype-specific microsatellite (SSR) markers. Eighteen sugarcane genotypes including 13 active cultivars and five elite QT-series clones bred locally were screened for genetic variability with 21 SSR primer pairs. All the primer pairs were highly polymorphic and amplified a total of 167 alleles with an average of eight alleles per primer pair. The average polymorphism information content (PIC) value was 0.86 with a range of 0.68 and 0.92. A UPGMA dendrogram categorized the 18 sugarcane genotypes into three major groups containing three, ten and five genotypes, respectively. No geographical affinity was observed among genotypes within the same group. Eight SSR primer pairs produced cultivar-specific alleles, of which five alleles were unique to the QT-series clones, namely, SMC334BS-165 and SMC851MS-145 in QT 08-558, mSSCIR43-229 in QT 4, SM597CS-182 in QT 08-536 and SMC7CUQ-168 in QT 06-212. The clone-specific SSR alleles will be useful in identifying elite QT-series clones for use in the sugarcane crossing programs in China.