Freshwater fish are a group that is especially susceptible to biodiversity loss as they often exist naturally in small, fragmented populations that are vulnerable to habitat degradation, pollution and introduction of exotic species. Relatively little is known about spatial dynamics of unperturbed populations of small-bodied freshwater fish species. This study examined population genetic structure of the purple spotted gudgeon (Mogurnda adspersa, Eleotridae), a small-bodied freshwater fish that is widely distributed in eastern Australia. The species is threatened in parts of its range but is common in coastal streams of central Queensland where this study took place. Microsatellite (msat) and mitochondrial DNA (mtDNA) variation was assessed for nine sites from four stream sections in two drainage basins. Very high levels of among population structure were observed (msat F(ST) = 0.18; mtDNA Φ(ST) = 0.85) and evidence for contemporary migration among populations was rare and limited to sites within the same section of stream. Hierarchical structuring of variation was best explained by stream section rather than by drainage basin. Estimates of contemporary effective population size for each site was low (range 28 - 63, Sibship method), but compared favorably with similar estimates for other freshwater fish species, and there was no genetic evidence for inbreeding or recent population bottlenecks. In conclusion, within a stable part of its range, M adspersa exists as a series of small, demographically stable populations that are highly isolated from one another. Complimentary patterns in microsatellites and mtDNA indicate this structuring is the result of long-term processes that have developed over a remarkably small spatial scale. High population structure and limited dispersal mean that recolonisation of locally extinct populations is only likely to occur from closely situated populations within stream sections. Limited potential for recolonisation should be considered as an important factor in conservation and management of this species.