Eurycoma longifolia, among the more precious medicinal herbs, has many promising benefits. However, the limited information on genetic diversity of the species has hindered many further related studies such as breeding programme. This study aims to evaluate the transferability of retrotransposon-based markers, Inter-Retrotransposon Amplified Polymorphism (IRAP) to evaluate the genetic diversity within and between two selected provenances. Thirteen IRAP markers were initially screened for their transferability properties. Findings showed that retrotransposon elements were present in the genome of E. longifolia. Four single primers and two combinations of primers were effectively utilized to analyse the genetic diversity of E. longifolia. Genetic diversity assessment indicated that the total diversity (H_T) was 0.2396 and the diversity within the population (H_S) was 0.2233. The coefficient of gene differentiation (G_ST) was 0.0680, signifying that there were 6.8% total genetic variations between the provenances and 93.2% variations among individual accessions within the provenances. Thus, the genetic variations between selected provenances were lower than the genetic variations within the provenances.