The fluvial-lacustrine and aeolian deposits in the Hetao Basin provide a record of regional palaeo-environment changes that can be useful for constraining the evolution of the palaeo-Yellow River. Here, we provide a new chronology for middle Pleistocene through Holocene sedimentary sequences in the Hetao Basin. Combined with new micropaleontological results and previous geochemical and biological data, this framework reconstructs the hydro-geological evolution of the Hetao Basin over the last 500 ka. Based on the available field data and water balance modeling, we suggest that the palaeolakes in the Hetao Basin developed at least similar to 450 ka ago, and subsequently expanded and contracted during interglacial and glacial intervals, respectively. The peak palaeolake area may have reached 34,000 km(2), covering most of the Hetao Basin, and would have been predominantly fed by the Yellow River. However, at similar to 128 ka, the lake overflowed due to a significant wetter and warmer climate, and afterwards the Yellow River became reintegrated, flowing out of the Hetao Basin, likely along a near-modern pathway. During this process, the palaeolake area shrunk by about 85 % and only remained in the western part of the Hetao Basin. This smaller palaeolake was still fed by a branch of the Yellow River until about 5 ka. Our results better constrain the late Quaternary hydro-geological history of palaeolakes in the Hetao Basin on glacial-interglacial timescales. These findings are not only important for reconstructing the evolution of the palaeo-Yellow River, but also for understanding regional aeolian dynamics and hydraulic erosion processes in the Chinese Loess Plateau.