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Abstract Background The identification of banana cultivars genome is needed to provide a valid identity from the accession of bananas which are used as basic data in the management of in situ and ex situ banana conservation as well as for further breeding of banana cultivars. Results The size of PCR-amplified matK ranged from 844 to 860 bp and showed a high variability. The haplotype diversity was 0.9048 with nine haplotypes. Haplotype distribution map revealed the lineage patterns of banana cultivars from Java. Reconstruction of genetic relationships using the maximum likelihood, maximum parsimony and Bayesian inference algorithms produces tree topologies and classifications that are grouped according to their genomic groups, into three main clades, i.e., AA/AAA, AAB and ABB. Based on the previously derived age constraints and fossil data, we estimate (Musaceae) that genetic divergence times of all samples occurred during the Eocene (95% HPD: 51.9 Mya), Musa acuminata group (AA, AAA, AAB) with Musa balbisiana group (BB and ABB) occurred during the Oligocene (95% HPD: 26 Mya), and the separation on each banana cultivars occurred during the Middle Miocene to Pliocene (95% HPD: 16.5–2.5 Mya). Conclusions From this study, we conclude that all studied cultivars are closely related according to its genomic groups with high variation. Genetic variation among those cultivars creates nine haplotypes. The development of variety which leads to the formation of different banana cultivars had suggested to be occurred long ago along with human migration and domestication.