Understanding population connectivity of marine species is becoming increasingly important for conservation planning, especially for species inhabiting coastal environments where anthropogenic threats are escalating. A recent acoustic tracking study on Port Jackson sharks (PJs) breeding in Jervis Bay, NSW, has discovered that PJs undertake yearly long-distance migrations as far south as Tasmania. There is also early evidence of PJs breeding in Sydney waters migrating south at similar times. However, not all sharks from Jervis Bay appear to follow the same the migration pattern and the reasons for these migrations are generally not well understood. Given the overlap of migration pathways between Jervis Bay and Sydney aggregations, we investigated the genetic structure and dispersal patterns of PJs from these two regions. We analysed 89 PJs samples using ten highly polymorphic microsatellites that were developed for this study as well the mitochondrial DNA (mtDNA) control region. Despite the breeding sites being separated by only 200km, significant population structure was detected using mtDNA. This result was not observed with microsatellites and further analysis based on assignment indices confirmed male-biased dispersal for this species. However, observations of fine-scale movement patterns in Jervis Bay over multiple breeding seasons have shown that adult males are generally more philopatric than females, returning to the same breeding site year after year. Taken together, these results suggest that juvenile male PJs may be dispersing from their natal area, thereby facilitating gene flow among these NSW coastal populations. This study emphasizes the need for multi-disciplinary approach to understanding population connectivity, especially when developing conservation management strategies for threatened or ecologically important marine species.