Non-perennial streams, which lack year-round flow, are widespread globally. Identifying the sources of water that sustain flow in non-perennial streams is necessary to understand their potential impacts on downstream water resources, and guide water policy and management. Here, we used water isotopes (δ18O and δ2H) and two different modeling approaches to investigate the spatiotemporal dynamics of young water fractions (Fyw) in a non-perennial stream network at Konza Prairie (KS, USA) during the 2021 summer dry-down season, as well as over several years with varying hydrometeorological conditions. Using a Bayesian model, we found a substantial amount of young water (Fyw: 39.1–62.6%) sustained flows in the headwaters and at the catchment outlet during the 2021 water year, while 2015–2022 young water contributions estimated using sinusoidal models indicated smaller Fyw amounts (15.3% ± 5.7). Both modeling approaches indicate young water releases are highly sensitive to hydrological conditions, with stream water shifting to older sources as the network dries. The shift in water age suggests a shift away from rapid fracture flow toward slower matrix flow that creates a sustained but localized surface water presence during late summer and is reflected in the annual dynamics of water age at the catchment outlet. The substantial proportion of young water highlights the vulnerability of non-perennial streams to short-term hydroclimatic change, while the late summer shift to older water reveals a sensitivity to longer-term changes in groundwater dynamics. Combined, this suggests that local changes may propagate through non-perennial stream networks to influence downstream water availability and quality.