Habitat fragmentation can have several adverse genetic impacts on populations. Assessing the extent of these threatening processes is essential in conservation management. In the present study, we investigated the genetic population structure of the endangered pygmy bluetongue lizard, Tiliqua adelaidensis, which is now restricted to a few small fragments of its previously more extensive grassland habitat. The aim of our study was to investigate genetic diversity and gene flow both among and within sample sites. The information will assist in making recommendations for habitat conservation and translocation programs. We collected DNA from 229 individuals from six isolated sample sites and genotyped them for 16 polymorphic microsatellite loci. Across all six sample sites, observed heterozygosity ranged from 0.75 to 0.82. There was no evidence of population bottlenecks and little evidence of inbreeding due to consanguineous mating. Genetic differentiation was low to moderate although significant for all pairs of sample sites (F_ST = 0.021–0.091). Results from Bayesian clustering analyses revealed distinct clusters in the overall sample and suggested restricted gene flow between sample sites separated by distances ranging from 1.7 to 71.6 km. By using spatial autocorrelation, we also found a significant genetic structure within sample sites at distances up to 30 m, suggesting restricted gene flow even in small patches of continuous habitat. It will be important to preserve this finely clustered population structure in captive breeding and translocation programs. Increasing opportunities for gene flow through habitat corridors or population augmentation may help maintain genetic diversity and prevent an increase in differentiation. Although endangered species do not always present model systems for studying fragmentation, our approach shows how important genetic information can be acquired to aid conservation in highly fragmented ecosystems.