Sugarcane (Saccharum officinarum L.) is an agricultural commodities with a great extent of diversity and high economic value. In Indonesia, the great extent of diversity of sugarcane is evidenced by a large number of cultivars cultivated. Sugarcane diversities at the molecular level can be seen using DNA barcodes, one of which is the matK. The purpose of the study was to identify and characterize matK and reconstruct the phylogenetic tree to determine the phylogeny of 24 sugarcane cultivars Indonesia. matK was amplified using the PCR method with matK F-5’ATGATTAATTAAGAGTAAGAGGAT-3’ and matK R-5’AATGCAAAAATTCGAAGGGT-3. Results showed that the matK gene was successfully amplified as many as 1531 bp. The sequencing process was done to determine the nucleotide sequence and compared with those of the GenBank database. It showed that the samples used had a similarity of 98.87%-99.44% to that of matK in Saccharum officinarum, Saccharum hybrid cultivar and Saccharum spontaneum. Reconstruction of the phylogenetic tree showed that the samples used were located in the same clade with a zero genetic distance, while all the references from NCBI were also located in the same clade. The analysis of genetic variation indicated that it had no haplotype value.