The effect of flooding on the dormancy level of Setaria parviflora seeds from a non-flooded upland and a seasonally (winter–spring) flooded lowland in the Pampa grasslands of Argentina was investigated. Seeds from both communities were subjected to reciprocal burial treatments in the two habitats, and exhumed during and after the flooding season. Effect of immersion in water at 5°C was compared to incubation of seeds on the surface of water-saturated paper at the same temperature. After exhumation of the buried seeds or immersion treatments, germination was assayed at 25°C and at 20/30°C in the dark or in combination with light. Burial in the lowland, which was flooded in winter–spring, significantly reduced germination, while burial in the non-flooded upland did not reduce germination. Similarly, immersion in water at 5°C significantly reduced germination compared to non-immersed controls. During summer, seeds buried in the lowland showed increased capacity to germinate, particularly when exposed to fluctuating temperatures or light. Thus, flooding induced secondary dormancy in S. parviflora seeds, and it was broken during the non-flooding season. These responses of the seeds would prevent germination until there was no further risk of flooding. Remarkably, in S. parviflora seeds harvested from both habitats, we observed essentially the same germination requirements after flooding. However, some slight differences were detected between the seed populations exhumed from the lowland site, indicating that flooding had larger effects on the seeds from the upland community. This suggests some differentiation of these populations evident only after flooding in the field.