Populations of marine mammals can show the signature of phylogeographical breaks and restricted connectivity despite the apparent lack of physical boundaries in the marine environment and their high dispersal abilities. Dugongs (Dugong dugon) do not appear to undertake regular migrations but may show localised movement related to water temperature or seagrass availability. Previous mitochondrial DNA studies suggested that despite a strong phylogeographic break in the Torres Strait, there is local panmixia in Australian waters. In this study, we conducted a comprehensive study of the four major dugong populations in southern Queensland. We analysed genotypes of 1,293 dugongs based on 24 microsatellite loci from the four major dugong locations in the region (from south to north): Moreton Bay, Great Sandy Straits, Hervey Bay and Shoalwater Bay. Diversity levels were similar across locations (observed heterozygosity 0.48–0.52, allelic richness 4.3–4.5). There was low but significant population differentiation in southern Queensland (FST ranged from 0.005 to 0.040 and Jost’s DEST ranged from 0.001 to 0.031 for microsatellite data). Bayesian clustering analysis implemented in STRUCTURE largely distinguished the southern Moreton Bay population from the three more northern populations. Twelve mitochondrial control region haplotypes identified from a subset of 182 samples confirmed significant population structuring (FST ranged from 0.16 to 0.28). These data suggested that the frequency and extent of dugong movements are insufficient to disrupt the long-term existence of at least two breeding populations in southern Queensland.