A primary factor in population management and wildlife conservation is the delineation of population units derived from descriptions of population genetic structure. Yet, predicting factors that influence the patterns of gene flow in a population particularly at landscape scales remains a major challenge in evolutionary biology. Here we report a population genetic study of the mud crab Scylla olivacea examined based on a 542 bp segment of the mitochondrial DNA cytochrome c oxidase I gene among 91 individuals from six localities in the west and east coast of Peninsular Malaysia. In total 55 unique haplotypes were distinguished with 45 private haplotypes and a single common haplotype shared among all populations studied. The other ten haplotypes were shared among various populations. The sharing of this haplotype reflects the connection of the mangrove areas between east and west coast of Peninsular Malaysia. High haplotype diversity (h = 0.968 ± 0.021; mean ± SD) and low nucleotide diversity (π = 0.120 ± 0.015; mean ± SD) were displayed, which may be indicative of genetic bottleneck events. No significant phylogenetic lineages were recognized using neighbour-joining and maximum parsimony methods. Hierarchical AMOVA analysis indicated that 99.33 % of the genetic variation was contained within populations and 0.67 % occurred among populations, suggesting no geographical patterning among populations studied, supported by F st test. Mismatch distribution analysis showed that the observed distribution of the pairwise mutation differences among haplotypes was multimodal, which is not concordant with a sudden range expansion scenario. However, neutrality tests showed non-significant negative values suggesting that the populations studied may have experienced past population growth, but the expansion may have been restricted to separate local areas that resulted in the non-significant negative Fu's Fs and Tajima's D value. Overall, this present preliminary study was able to be a reference on the phylogenetic relationships and assessment of genetic structure of Scylla sp. in Malaysia.