Banana is one of the most essential commodities in Bali island. It is not only for nutrition sources but also for cultural and religious aspects. However, Bali banana genetic diversity has not been explored; therefore, in this study, we focused on its genetic relationship using a molecular approach. This research aimed to determine the genetic relationship of Bali banana cultivars using the internal transcribed spacer 2 (ITS-2) region as a molecular marker. A total of 39 banana samples (Musa spp. L.) were collected from Bali island. The ITS-2 DNA regions were then amplified and sequenced from both ends. ITS-2 sequences were predicted using the ITS2 Database (http://its2.bioapps.biozentrum.uni-wuerzburg.de/). The multiple sequences alignment was performed using ClustalX for nucleotide-based tree and LocARNA to provide the secondary structure information. Phylogenetic trees were constructed using neighbor-joining (Kimura-2-parameter model, 1,000 bootstrap). The result showed that two clades were formed, one clade was abundant in A genome (AA and AAA), and the other rich in the B genome (BB and ABB). This result suggested that cultivars that had similar genomic compositions tended to be grouped within the same clade and separated with different genomic compositions. This study gives perspectives that ITS-2 sequences in bananas are quite similar and differ much compared to other families. Secondary structure has been described to provide more robust resolving power in phylogenetic analysis.