Germplasm innovation can provide materials for breeding sugarcane cultivars. Saccharum officinarum is the main source of high-sugar and high-yield genes in sugarcane breeding. ‘Nobilization’ is the theoretical basis for exploiting S. officinarum, and S. officinarum authenticity directly affects sugarcane nobility breeding efficiency. Herein, the authenticity of 22 SLC-series S. officinarum clones imported from Sri Lanka and preserved in the China National Germplasm Repository of Sugarcane (NGRS) was explored by four-primer amplification-arrested mutation PCR (ARMS PCR) and somatic chromosome number counting. The amplified bands from SLC 08 120 and SLC 08 131 were the same with those from S. officinarum clone Badila, i.e. a common band of 428 bp and a S. officinarum-specific band of 278 bp, hence they were tentatively assigned as S. officinarum clones. The other 20 SLC clones had both 278 bp (S. officinarum-specific) and 203 bp (S. spontaneum-specific) bands, which are hybrid characteristics. In addition, the chromosome numbers of SLC 08 120 and SLC 08 131 are both 80, belong to typical S. officinarum. While the chromosome numbers of the other 20 materials are ranging from 101 to 129, consistent with hybrids of S. officinarum and S. spontaneum. This molecular cytological characterization indicates that among the 22 introduced SLC-series clones, only two, SLC 08 120 and SLC 08 131, were S. officinarum. Future agronomic trait and resistance analyses could facilitate their use as crossing parents in sugarcane breeding.