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Aim Great Artesian Basin (GAB) springs in central Australia support several plant species otherwise not found in the arid zone. Evolutionary theory predicts that isolated populations will experience reductions in gene flow and genetic diversity, and higher levels of inbreeding. Our aim was to test this prediction by comparing the genetic structure of cutting grass, Gahnia trifida, (Cyperaceae) on disjunct GAB springs with coastal populations that have experienced recent fragmentation. Location Naturally isolated GAB springs near Lake Eyre, central Australia, and coastal sites in southern Australia. Methods We used 13 microsatellite markers to genotype 267 samples from six GAB spring and four coastal G. trifida populations. These data were used to estimate population genetic statistics and contemporary and historical measures of gene flow in the two regions. Results GAB spring populations display lower levels of genetic diversity compared with coastal populations. Furthermore, GAB spring populations displayed much higher levels of genetic differentiation (FST=0.52) than populations at coastal sites (FST=0.22). Several coastal populations exhibited historical genetic connectivity, whereas analysis of molecular variation (AMOVA) and contemporary migration rate estimates indicate that populations from GAB spring groups are demographically independent. Main conclusions Divergence estimates based on microsatellite data suggest restriction of central Australian G. trifida populations to refugial spring habitats since at least 1528ka, a period that spans the Last Glacial Maximum. Dispersal amongst spring groups is insufficient to counteract the effect of genetic drift, leading to a loss of genetic diversity. Species persisting in isolated or fragmented habitats are likely to suffer adverse effects on genetic traits, potentially increasing their risk of extinction.