The existence of Hesperian age (3.7–3.4 Ga) surface water bodies on Mars is a contentious issue, often conflicting with favored climate models. Extensive lakes are proposed to have filled parts of Valles Marineris during this period, yet evidence for their presence and temporal continuity is poorly constrained. Here we report geomorphic and chronologic evidence for the initiation and demise of a voluminous lake system within the basins of eastern Valles Marineris. We find that independent, kilometer-deep lakes were present here well after the wetter, global climate optimum that characterized the previous Noachian epoch (4.1–3.7 Ga). Relative and impact crater chronologies of flood channels emerging from lake basins indicate relatively late lake spillover in the Early Amazonian (ca. 3.0 Ga). Drawdown of the lake and cessation of interbasin sedimentation may be recorded by a similar Early Amazonian (ca. 3.1 Ga) crater retention age on the surface of Capri Mensa, a 4-km-tall, sulfate-bearing interior layered deposit. The topography data demonstrate that incision of the bedrock barriers between the basins during spillover was driven by a dramatic local base-level difference between the lake surface and downstream basin floors. We postulate that the lake spillover process created an integrated drainage routing system between a voluminous equatorial water supply and the northern plains basin.