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High-elevation endorreic lakes in the Southern Altiplano of South America represent a major source of local biodiversity. Size and depth of wetlands in Northwest Argentine (NWA) and Southwest Bolivia (SWB) have shown to be very sensitive to basin hidrological balances, and consequently, very vulnerable to deleterious effects from climate changes. The management of these water resources requires a comprehensive knowledge of their natural variability over multiple time scales. In this study we present a multi-century reconstruction of past lake-area fluctuations in the NWA and SWB, inferred from Polylepis tarapacana tree-ring records. Between 1975 and 2009 interannual lake area fluctuations from nine lakes were quantified based on Landsat satellite images. A composite P. tarapacana tree-ring chronology was developed. Correlations analyses were performed to screen potential predictor tree-ring chronologies for reconstruction models. Inter-annual lake area fluctuations were positively correlated with inter-annual variations of the radial growth of P. tarapacana. A tree-ring chronology (601 years long) was use as predictor, in a regression model, to reconstruct the annual (January–December) mean lake area from nine endorreic lakes. The chronology captures 60% of the total variance in lake-area fluctuations and shows adequate levels of cross-validation. The twentieth century was unusual in the long-term context provided by the reconstruction; a persistent negative trend in lake area is clear in the reconstruction during the past century and is consistent with glacier retreat and other climate proxies from the Altiplano and tropical Andes. These results provide a baseline for the historical range of variability in lake fluctuations, and thus should be considered for the management of biodiversity and water resources in the region, particularly in relation to future XXI century climate scenarios.