Both present-day and past processes can shape connectivity of populations. Pleistocene vicariant events and dispersal have shaped the present distribution and connectivity patterns of aquatic species in the Indo-Pacific region. In particular, the processes that have shaped distribution of amphidromous goby species still remain unknown. Previous studies show that phylogeographic breaks are observed between populations in the Indian and Pacific Oceans where the shallow Sunda shelf constituted a geographical barrier to dispersal, or that the large spans of open ocean that isolate the Hawaiian or Polynesian Islands are also barriers for amphidromous species even though they have great dispersal capacity. Here we assess past and present genetic structure of populations of two amphidromous fish (gobies of the Sicydiinae) that are widely distributed in the Central West Pacific and which have similar pelagic larval durations. We analysed sections of mitochondrial COI, Cytb and nuclear Rhodospine genes in individuals sampled from different locations across their entire known range. Similar to other Sicydiinae fish, intraspecific mtDNA genetic diversity was high for all species (haplotype diversity between 0.9-0.96). Spatial analyses of genetic variation in Sicyopus zosterophorum demonstrated strong isolation across the Torres Strait, which was a geologically intermittent land barrier linking Australia to Papua New Guinea. There was a clear genetic break between the northwestern and the southwestern clusters in Si. zosterophorum (?ST = 0.67502 for COI) and coalescent analyses revealed that the two populations split at 306 Kyr BP (95% HPD 79-625 Kyr BP), which is consistent with a Pleistocene separation caused by the Torres Strait barrier. However, this geographical barrier did not seem to affect Sm. fehlmanni. Historical and demographic hypotheses are raised to explain the different patterns of population structure and distribution between these species. Strategies aiming to conserve amphidromous fish should consider the presence of cryptic evolutionary lineages to prevent stock depletion. � 2013 Taillebois et al.