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Aim The conservation of plant species biodiversity has been identified as a crucial factor for the resilience of dryland ecosystems in the face of climate change and desertification. Duma florulenta (lignum) is a keystone species that facilitates biodiversity in the floodplains and wetlands of Australia's dryland river systems. This paper explores spatial genetic structure of lignum and investigates factors influencing dispersal and gene flow within and among river catchments of the northern Murray-Darling Basin. Location Northern Murray-Darling Basin, eastern Australia. Methods A total of 122 individual plants from subpopulations located on rivers in four adjacent catchments were genotyped using 10 microsatellite markers. Microsatellite data were then analyzed using population genetic techniques to evaluate levels of gene flow and genetic structure and identify factors influencing dispersal. Results Results suggest high levels of gene flow between lignum subpopulations of the northern Murray-Darling Basin. AMOVA revealed small but significant differences between subpopulations, and STRUCTURE analysis did not detect meaningful structure when sampling information was not provided. However, when sampling information was supplied using the LOCPRIOR model, three genetic clusters were identified. All Lower Balonne subpopulations were assigned to cluster 1 while a number of the other subpopulations showed mixed ancestry. Weak relationships were identified between pairwise genetic distance and geographic as well as river distance, although the R-2 value of the former was only half that of the latter. Main conclusions Patterns of genetic variation suggest frequent long-distance overland gene flow largely as a result of the movement of seeds via floodwater. Therefore, maintenance of natural variability in flow regime is key both to maintain conditions favorable to recruitment and to promote dispersal and gene flow across the landscape. However, given future climate change projections persistence may be more reliant on the species ability to endure long periods of drought between flood events.