The Santiago–Guadalajara River Basin has an area of 10,016.46 km². The Metropolitan Area of Guadalajara, within the basin, is the second-largest city in the country, with more than 5 million inhabitants. The growth of the urban population, as well as industrial and agricultural activities with insufficient infrastructure for the sanitation of wastewater and its reuse, have caused environmental deterioration of surface waters and gradual depletion of groundwater resources. To assess the level of contamination in surface waters from the presence of heavy metals in the basin, a monthly monitoring campaign was carried out at 25 sampling stations located in the main and tributary streams from July 2021 to April 2022. The following decreasing sequence was found according to the mean concentration values: Fe > Al > Mn > B > Ba > Zn > As > Cu > Cr > Ni > Pb > Cd. The Heavy Metal Pollution Index (HPI) method was applied to assess the level of risk to aquatic life, finding an average global HPI value of 305.522 for the basin, which classifies it as in the critical contamination range.