The Brazilian Cerrado biome provides relevant ecosystem services for Brazil and South America, being strategic for the planning and management of water resources as well as for agribusiness. The objective was to evaluate the water quality along the course of the Córrego da Formiga in a virgin portion of the Brazilian Cerrado, the relationship of land use with physical-chemical and biological parameters of the water, and the inflow of the tributary. Five water collection points were defined (between the source and mouth) and observed on a quarterly scale in 2015, water samples were collected and analyzed for physical-chemical and biological parameters in the laboratory, and flow measurements were performed at the same point and day of water collection. To identify and quantify land use and land cover (LULC) in the watershed, an image from the Landsat8-OLI satellite was obtained, and other geomorphological data from hypsometry (Topodata-INPE) were obtained to generate the slope, basin delimitation, and contribution area for each water collection point. The LULC percentages for each area of contribution to the water collection points were correlated with the physical-chemical and biological parameters of the water and submitted to multivariate analysis (PLS-DA) for analysis and grouping among the five analyzed points. Changes in water-quality patterns were more pronounced concerning the time when the first and last sampling was performed (rainy period) and may be influenced by the increase in the volume of water in these periods. The stream flow is highly variable over time and between points, with the lowest recorded flow being 0.1 L s−1 (P1) and the highest being 947.80 L s−1 (P5). Córrego da Formiga has class III water quality (CONAMA resolution 357), which characterizes small restrictions on the use of water for multiple uses. The soil cover with native vegetation is just over 12%, while the predominance was of the classes of sugar cane (62.42%) and pasture (19.33%). The PLS-DA analysis allowed separating the water analysis points between P1, P2, P3, and P5, while P4 was superimposed on others. It was also possible to verify that the parameters that weighed the most for this separation of water quality were pH, alkalinity_T, alkalinity_h, calcium, and hardness, all with a tendency to increase concentration from the source (P1) to the mouth (P5). As for water quality, it was also possible to verify that points P2 and P5 presented better water-quality conditions.
