A multidecadal-scale lake-level reconstruction for Lago Wiñaymarca, the southern basin of Lake Titicaca, has been generated from diatom species abundance data. These data suggest that ~6500 cal yr BP Lago Wiñaymarca was dry, as indicated by a sediment unconformity. At ~4400 cal yr BP, the basin began to fill, as indicated by the dominance of shallow epiphytic species. It remained somewhat saline with extensive wetlands and abundant aquatic plants until ~3800 cal yr BP, when epiphytic species were replaced by planktic saline-indifferent species, suggesting a saline shallow lake. Wiñaymarca remained a relatively shallow lake that fluctuated on a multidecadal scale until ~1250 cal yr BP, when freshwater planktic species increased, suggesting a rise in lake level with a concomitant decrease in salinity. The lake became gradually fresher, dominated by deep, freshwater species from ~850 cal yr BP. By ~80 cal yr BP, saline-tolerant species were rare, and the lake was dominated by freshwater planktic diatoms, resembling the fresh and deep lake of today. These results reveal a more dynamic and chronologically specific record of lake-level fluctuations and associated ecological conditions that provide important new data for paleoclimatologists and archaeologists, to better understand human-environmental dynamics during the mid- to late Holocene.