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Pasak bumi (Eurycoma longifolia Jack) is a shrubs growing wildly in the forests of Southeast Asia and widely used throughout the region because of its medicinal properties. Uncontrolled harvesting of wild-grown trees has led to rapid decrease of it natural populations, as well as causing a potential decrease in genetic diversity. Information about population genetic of pasak bumi still not determined yet, including the optimal DNA isolation and it reference marker. Therefore, our research was conducted to get information about optimal DNA isolation using CTAB methods and reference marker for population genetic study. DNA isolation was conducted through CTAB (Cetyl Trimetyl Ammonium Bromide) method. The yield DNA was PCR amplified using four barcoding standard marker those were ITS, matK, rbcL and trnL-trnF. The result showed that CTAB method was able to yield good quality DNA and 30% dilution produced the best band. The trnL-trnF primer was able to amplified DNA of pasak bumi with 50°C annealing temperature, rbcL with 55°C annealing temperature and ITS with 50°C annealing temperature. While the matK primer failed to amplified. [ABSTRACT FROM AUTHOR]