A quantitative study of toxicity levels of the San Pedro River and its main tributaries around the city of Aguascalientes, Mexico was conducted. Our study determined individual CL(50) values for each sampling point at 3 different times of the year corresponding to the main seasons of the year in terms of the hydrological cycle (dry, low rain and high rain season). Those LC(50) values were used to calculate the acute. Toxicity Units (aTU) that allowed us to compare levels of toxicity along the San Pedro River and two of its main tributaries. The sample that showed highest toxicity was IPIVA. This is due to the large quantity of industrial discharges that receives. Its effluent was responsible for the largest contribution of toxicity to the San Pedro River over the three rounds of sampling of this study. Our study classified an important portion of the San Pedro River and two of its main tributaries in toxic, moderately toxic and lightly toxic. No portion of the river studied was free of toxicity, either acute or sublethal. This study demonstrated that in spite of the operation of several water treatment plants along the San Pedro River, for the most part, the water quality of the river is still unacceptable.