South Kalimantan on the Borneo Island is one of the center origin and diversity of durian (Durio spp.) germplasm in Indonesia. This study was conducted to investigate the genetic diversity and relationships of local durian (Durio zibethinus Murr.) cultivars from South Kalimantan’s Province by RAPD markers. Eleven plant samples of local durian cultivars trees and five selected RAPD primers, namely OPA-01, OPA-02, OPA-07, OPA-16, OPA-18, and OPA19 were used. DNA samples were extracted from leaf tissues according to the protocol of Nucleon Phytopure (Amersham Bioscience). Amplifications were performed on a PCR Thermal Cycler (Applied Biosystem 9700). Variation of the PCR products of each primer was scored based on the presence (1) and absence (0) of bands on the images taken from the gels. The NTSys-PC software was used to compute Dice’s coefficients of similarity, and the dendrogram was constructed using UPGMA method. Results showed that 11 local durian cultivars of South Kalimantan’s Province have high diversity which indicated by polymorphism degree of 82.17% and clustering analysis where those cultivars were grouped into six distinct clusters. Based on the result, durian ‘Likol’ had closely related to the durian ‘Sipisang’ and ‘Sihabuk’ at the level 95% of similarity, while durian ‘Enam Hapat’ had separated at a distance from others. This information be used as a consideration in conservation and management improvement strategies (breeding program) of the durian in Indonesia.