Present study investigates the genetic diversity and genetic distribution of the longtail tuna Thunnus tonggol collected from east Malaysia (Borneo states of Sabah and Sarawak) based on mitochondrial DNA D-loop sequence analysis. 58 fish samples were obtained, specifically from Kota Kinabalu, KK (n = 22), Miri, MR (n=20) and Bintulu, BT (n = 17). DNA template was isolated using the salt extraction method. Final length of 404 base pair (bp) D-loop sequences revealed 52 haplotypes that comprise of 77 variable sites (38 of parsimony informative and 39 singleton). A total of 20 haplotypes were found in KK, 19 haplotypes in MR and 16 haplotypes in BT. Molecular diversity indices revealed high haplotype diversity and low nucleotide diversity in all populations; KK (h = 0.9913 ± 0.0165, π = 0.00239 ± 0.0127), MR (h = 0.9942 ± 0.0193, π = 0.0226 ± 0.0121) and BT (h = 0.9926 ± 0.0230, π = 0.0196 ± 0.0171). Population comparison pairwise FST show that KK and BT were significantly genetically differentiated. The result from this study will be beneficial for fisheries management and also to provide information on the population genetics of T. tonggol in East Malaysian waters.