Eurycoma longifolia (Simaroubaceae) is a popular medicinal plant from the South-East Asian rainforest that often used as an aphrodisiac and anti malaria. The main supplies of E. longifolia from Indonesia are from Sumatra and Kalimantan. The increasing demand of this species for medinical industries has led to illegal export in Indonesia. This study was aimed to identify specific nucleotides as molecular identity of E. longifolia from Sumatra based on DNA sequences of trnL-F plastid gene. Twenty four samples of E. longifolia were collected from three populations in Sumatra to be sequenced. The aligned sequence of the trnL-F_is 960 bp, and is an A/T rich region (A: 30.0%, T: 34.3%, C: 16.8% and G: 18.8%). The homology search using BLASTn of the genbank NCBI showed that the nucleotide composition of E. longifolia used in this study was similar to E. longifolia MH751519.1 (100%) and KP995919.1 (99.76%), Simaba morettii MG599450.1 (99.76%), and E. apiculata GU593014.1 (99.76%). Close examination of the gene structure and composition showed that the DNA sequences or E. longifolia from Sumatra have five nucleotides variations that did not possesed by the reference E. longifolia from the genbank. These variations comprised four base substutions and one insertion-deletions event, and were occurred mostly in the trnL intron region. When a phylogenetic analysis was perfomed, its results confirmed the correct identity of the samples species by their position was at the same clade as the other E. longifolia. The results from this study is expected to assist identification of herbal medicine containing E. longifolia from Sumatra, Indonesia.