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Flood tolerance is crucial to the survival of tree species subject to long periods of flooding such as those present in the Amazonian Varzea. Tolerance can be mediated by adjustments of metabolism, physiology and morphology, reinforcing the need for investigation of the physiological and biochemical mechanisms used by tropical tree species to survive this stress. Moreover, such mechanisms may vary between populations that are subjected to differences in the frequency of flooding events. Here, we therefore aimed to identify the mechanisms used by two populations of the tropical tree Guazuma ulmifolia (Lam.) to tolerate flooding; an Amazonian population frequently exposed to flooding and a Cerrado population, adapted to a dry environment. Young plants were subjected to flooding of the roots and lower stem for 32 days, followed by 17 days of recovery. Amazonian plants exhibited greater increases in shoot length and higher maximum photosynthetic rate (Amax) compared to non-flooded plants from 7 days of flooding onwards, whilst increased Amax occurred later in flooded Cerrado plants and was not accompanied by increased shoot length. Lactate accumulated in roots of Cerrado plants after 24 h flooding, together with transcripts coding for lactate dehydrogenase in roots of both Cerrado and Amazonian plants. After 7 days of flooding lactate decreased and alcohol dehydrogenase activity increased transiently, together with concentrations of alanine, GABA and succinate, indicating activation of metabolic processes associated with low oxygen availability. Other amino acids also increased in flooded Cerrado plants, revealing more extensive metabolic changes than in Amazonian plants. Wetland and dryland populations of Guazuma ulmifolia revealed great capacity to tolerate flooding stress through a suite of alterations in photosynthetic gas exchange and metabolism. However, the integrated physiological, biochemical and molecular analyses realized here indicated that wetland plants acclimatized more efficiently with increased shoot elongation and more rapid restoration of normal metabolism.