Fine-scale genetic structure was investigated in three regional populations of the long-nosed potoroo (Potorous tridactylus) a threatened endemic marsupial. Two populations were from the Australian mainland and one from an island. Populations were sub-sampled at two sites, 6–8 km apart, connected by suitable habitat for dispersal. Factors influencing fine-scale structure were investigated by genotyping 157 individuals at 10 microsatellite loci and sequencing a ~621 bp region of the mtDNA control region. Results indicated that P. tridactylus populations exhibit significant intra-population structure, with significant F ST and Φ ST values recorded between subpopulations. This structure appeared mediated by small neighbourhood size, female philopatry and limited dispersal over 6–8 km, predominantly by males. Results highlighted several important features of P. tridactylus populations that have implications for conservation. Firstly, the small neighbourhood size suggests any investigations of intra-population structure should be conducted on a finer scale (e.g. 25–50 m) than many current monitoring programs. Secondly, the island populations were genetically depauperate, which may reflect processes occurring in many isolated ‘mainland island’ populations. Thirdly, the lower gene flow identified between populations separated by anthropogenically modified habitat suggests P. tridactylus is sensitive to changes in habitat configuration.