Regional teleconnections permit cross-continental modeling of hydroclimate throughout the world. Tree-rings are a good hydroclimatic proxy used to reconstruct drought and streamflow in regions that respond to common global forcings. We used a multi-species dataset of 32 tree-ring width chronologies from Chile and Uruguay as a climate proxy to infer annual streamflow (Q) variability in the Negro River basin, a grassland-dominated watershed of lowland Southeastern South America. A positive linear correlation between tree-ring chronologies from Central Chile and annual Negro River instrumental streamflow from 1957 to 2012 indicated a cross-continental teleconnection between hydroclimate variability in Central Chile and Northeastern Uruguay. This relationship was mediated in part by the El Niño Southern Oscillation (ENSO), whereby the El Nino 3.4 Index was positively correlated with regional rainfall, annual tree growth, and Q anomalies. Despite the proximity of Uruguayan tree-ring chronologies to Negro River hydrometric stations, the Chilean tree-ring chronologies best predicted annual streamflow. Thus, using tree-ring data from four long-term moisture-sensitive chronologies of the species Cryptocarya alba in Central Chile (32–34°S), we present the first streamflow reconstruction (1890–2009) in the lower La Plata Basin. The reconstruction supports regional evidence for increasing frequency of extreme flood years over the past century in Uruguay. We demonstrate how climate teleconnections that mediate local hydroclimate variability permit the cross-continental reconstruction of streamflow, filling a major geographical gap in historical proxies for flooding and drought in grassland biomes of the southern hemisphere.