Viola sp. (2n= 2x= 20) is the largest genus within the violet family, commonly known as medicinal plants due to its extensive use in traditional medicine. Recently, the cyclotide compounds found in the violet family have attracted considerable attention. Cyclotides are stable plant proteins with a circular peptide backbone crosslinked by a cystine knot of disulfide bonds, which can be used as scaffolds in drug design. The types of cyclotides are directly influenced by the kind of species and ecotypes, making it essential to maintain diverse genotypes of Viola species. Furthermore, assessing genetic variation aids researchers in utilizing plants for various purposes and helps prevent their extinction. The advancement of biotechnology, combined with other life sciences like bioinformatics, has increasingly highlighted the role of markers in identifying hereditary differences in DNA sequences. The iPBS molecular marker (Inter Primer Binding Sites) is one of the markers that amplifies repetitive mobile sequences (retrotransposons). The iPBS molecular marker was used as a method for identifying LTR sequence diversity and directly observing polymorphism among different plant varieties. There is no report of using this marker in the violet family so far. Therefore, this study aims to use iPBS marker to identify the genetic diversity among different ecotypes of violets.