A high resolution systematic geochemical mapping at regional-scale is considered to be the best available method to estimate geochemical baseline levels of trace elements in stream water, and is an essential part of environmental risk assessment. This methodology was applied in a project in the Itacaiúnas River Basin, southeastern Amazon, Brazil, which includes several mines of the Carajás Mineral Province (as part of the Itacaiúnas Geochemical Mapping and Background Project, ItacGMBP). A total of 1429 samples (including 55 duplicates) were collected in 2017 at 900 sites at one sample per microbasin, during both dry and rainy periods. The analyses of 34 elements were carried out using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). In general, the waters are slightly alkaline and are classified as mixed Ca-Na-HCO3 type, indicating that they are mainly influenced by silicate rock weathering. Most metal concentrations in the water are low, except Fe and Mn. Seasonality explains differences in metal concentrations, with higher values being obtained in the rainy season. Baseline threshold values (BTVs) were calculated separately for both seasons by employing different statistical methods: iterative 2σ and DF preferentially delivered a more restrictive or conservative levels, which can be represenative of the natural BTVs (NBTVs), considering as the least degraded with low or no significant level of anthropogenic influence; and 98th percentile provides the ambient BTVs (ABTVs), which consists of natural plus diffuse anthropogenic input in the defined area. The ABTVs of Fe and Mn significantly exceed the WHO (1998) and CONAMA 357/05 limits. Spatial distribution indicates that Fe and Mn are not strictly related to geologic setting, rather they are highly influenced by specific local land use as well as deep weathering of the catchment and intense leaching and run-off during the rainy season. However, higher Mn occurrence in the dry period results from redox cycling of Fe and Mn via biogeochemical processes. The ABTVs of Ni, Cr, and V are controlled by bedrock lithology (geologic setting), mainly associated with mafic-ultramafic rocks; Cu is associated with two large hydrothermally mineralized copper belts. The estimation of baseline levels of As, Se, and Pb were highly limited due to of the large number of samples with results less than detection limits, in this case the 95th percentile was used for their BTVs. Geochemical data as well as anomalous values for most of the metals indicate that anthropogenic influence from point sources is highly negligible in the basin, except at a few points, where high NO3- has been observed, probably due to more intense human and livestock activity. This study demonstrates that site-specific geochemical baseline assessment is a crucial factor when evaluating surficial water conditions in a large basin.