The phylogeny and biogeography of the Malayan freshwater crab genus Johora was studied using two mitochondrial genes, 16S rRNA (560 bp) and cytochrome oxidase subunit I (COI) (616 bp), and one nuclear gene, histone 3 (H3) (328 bp). Johora is shown to be monophyletic and composed of three clades that correspond with the topography of the Malay Peninsula. The three clades were estimated to be of similar age (c. 11 million years ago (mya)). The Malayan island of Pulau Tioman (with five species) was determined to have been colonised independently by two separate clades (at c. 11 and 5 mya, respectively), one of which evolved semiterrestrial habits, possibly in response to competition by the second. A partitioned Bremer support (PBS) analysis reveals that most of the support for the phylogenetic tree comes from the COI gene fragment and that the nuclear protein-encoding genes H3 is useful for reconstructing the relationships of Johora.