Periodic hydrological connectivity among fragmented floodplain habitats governs the persistence of aquatic biota. In dryland rivers, unpredictable flooding interspersed with low flows and drying drive their "boom and bust" ecology. During drying, aquatic habitats contract and fragment. Flooding connects and expands habitats, triggering productivity booms in waterbirds, fish, and plants. Microinvertebrates form the base of the food web and also flourish after floods, but their colonization pathways remain unknown.To determine the relative contribution of floodwater-transported propagules vs. emergence of in situ resting stages from inundated sediments, we studied short-term recruitment of microinvertebrates after flooding in the dryland Darling River, Australia. Lakes open to transported and emerging in situ microinvertebrates were compared with lake and laboratory microcosms closed to transported microinvertebrates. Floods imported most early colonists to floodplain lakes. In situ emergence from sediments was less important until weeks later but potentially sustained production over a longer period of inundation. Some taxa primarily colonized lakes via emergence whereas others were only transported to lakes. A spatially nested ANOVA approach spanning patches within lakes to among reaches revealed contrasting variability across hierarchical scales for taxonomic richness and densities of microinvertebrate taxa. This was most marked at the reach scale, possibly reflecting seasonal patterns in flooding. Microinvertebrate colonization sequences in the Darling River floodplain rely on connectivity between habitats during flooding. Ecological connectivity was greater at small spatial scales (patches within lakes 100–1000 m, among lakes 104–105 m) than at larger spatial scales (reaches 105–106 m). The strength of connectivity is likely to vary across spatial scales with the magnitude, duration, and rate of rise and fall of floods.