Gene flow is a key evolutionary driver of spatial genetic structure, reflecting demographic processes and dispersal mechanisms. Understanding how genetic structure is maintained across a landscape can assist in setting conservation objectives. In Australia, floodplains naturally experience highly variable flooding regimes that structure the vegetation communities. Flooding plays an important role, connecting communities on floodplains and enabling dispersal via hydrochory. Water resource development has changed the lateral-connectivity of floodplain-river systems. One possible consequence of these changes is reduced physical and subsequent genetic connections. This study aimed to identify and compare the population structure and dispersal patterns of tangled lignum (Duma florulenta) and river cooba (Acacia stenophylla) across a large inland floodplain using a landscape genetics approach. Both species are widespread throughout flood prone areas of arid and semiarid Australia. Tangled lignum occurs on floodplains while river cooba occurs along rivers. Leaves were collected from 144 tangled lignum plants across 10 sites and 84 river cooba plants across 6 sites, on the floodplain of the lower and mid Lachlan River, and the Murrumbidgee River, NSW. DNA was extracted and genotyped using DArTseq platforms (double digest RADseq). Genetic diversity was compared with floodplain-river connection frequency, and genetic distance (FST) was compared with river distance, geographic distance and floodplain-river connection frequency between sites. Genetic similarity increased with increasing floodplain-river connection frequency in tangled lignum but not in river cooba. In tangled lignum, sites that experience more frequent flooding had greater genetic diversity and were more genetically homogenous. There was also an isolation by distance effect where increasing geographic distance correlated with increasing genetic differentiation in tangled lignum, but not in river cooba. The distribution of river cooba along rivers facilitates regular dispersal of seeds via hydrochory regardless of river level, while the dispersal of seeds of tangled lignum between patches is dependent on flooding events. The genetic impact of water resource development may be greater for species which occur on floodplains compared with species along river channels.