Summary The dissolved organic matter (DOM) biogeochemistry of intermittent rivers is affected by periods of disconnection and drying after cessation of surface flow, with a pronounced concentration of solutes as pools evaporate and contract. However, inputs of alluvial ground water to stream pools may offset the effects of evaporation and influence the quantity and composition of DOM. We used δ¹⁸O and δ²H values of surface water and alluvial ground water (AW) together with DOM fluorescence excitation–emission spectroscopy to investigate changes in DOM biogeochemistry of pools from four intermittent streams in semi‐arid north‐west Australia. Our objective was to determine whether the fluorescence characteristics of DOM in pools are driven by their connectivity to alluvial ground water. Pool water δ¹⁸O values ranged from −8 to 13.1 ‰ and showed progressive enrichment as pools became disconnected from AW and subsequently evaporated. Parallel factor analysis of DOM fluorescence revealed that DOM in pools was generally dominated by humic‐like components, with lesser contributions from tryptophan‐like and tyrosine‐like components. DOC concentrations were higher in more evaporated pools. Humic‐like and tryptophan‐like DOM were also highest in pools isolated from AW, suggesting that inputs of AW had a dilution effect while also moderating evaporative concentration of solutes. In contrast, tyrosine‐like DOM was only weakly correlated with the hydrological status of pools. The proportion that each component contributed to total DOM fluorescence remained mostly consistent across both AW‐connected and evaporating pools. However, some pools with AW throughflow had higher contributions from tyrosine‐like fluorescence and lower contributions from humic‐like fluorescence. Disconnection from AW and subsequent evaporative concentration of solutes is an important control on DOM fluorescence characteristics in these intermittent streams. However, production and removal of DOM were only rarely influenced by AW connectivity, likely reflecting the high hydrological variability of intermittent rivers, even following cessation of surface flow.