Australian dryland rivers have distinctive ecologies, intimately linked to their variable flows. The contrasting states of flood and drought mean that dryland rivers fluctuate between being highly connected (during floods: low fragmentation) to being highly disconnected (during droughts: high fragmentation) with the degree of connection between waterbodies on a spatial scale and the time since last connection on a temporal scale strongly influencing community composition. Flow regulation reduces the frequency and duration of flooding, thereby decreasing flow variability and imposing stable low flow conditions and high connectivity between waterbodies. Using macroinvertebrate assemblage data from four Australian dryland rivers (River Murray, Darling River, Cooper Creek and Diamantina River), we calculated various indices of flow variability from 30-year hydrographs and assessed how well long-term flow variability, and therefore variable levels of habitat connection, could explain broad patterns of macroinvertebrate assemblage composition. Where hydrological connection between sites was strong (River Murray pool sites) there was extreme similarity between assemblages at each site. Also, for sites where hydrological disconnection was extreme there was similarity between assemblages. There were strong associations between the complex measures of flow variability and the assemblage composition of the four rivers, suggesting that flow variability, and therefore, variable levels of habitat connectivity, may be strong determinants of broad-scale assemblage composition in dryland rivers.