A comprehensive assessment of pesticides in surface waters is challenging due to the large number of potential contaminants. Most scientific studies and routine monitoring programs include only 15–40 pesticides, which leads to error-prone interpretations. In the present study, an extensive analytical screening was carried out using liquid chromatography–high-resolution mass spectrometry, covering 86% of all polar organic pesticides sold in Switzerland and applied to agricultural or urban land (in total 249 compounds), plus 134 transformation products; each of which could be quantified in the low ng/L range. Five medium-sized rivers, containing large areas of diverse crops and urban settlements within the respective catchments, were sampled between March and July 2012. More than 100 parent compounds and 40 transformation products were detected in total, between 30 and 50 parent compounds in each two-week composite sample in concentrations up to 1500 ng/L. The sum of pesticide concentrations was above 1000 ng/L in 78% of samples. The chronic environmental quality standard was exceeded for 19 single substances; using a mixture toxicity approach, exceedances occurred over the whole measurement period in all rivers. With scenario calculations including only 30–40 frequently measured pesticides, the number of detected substances and the mixture toxicity would be underestimated on average by a factor of 2. Thus, selecting a subset of substances to assess the surface water quality may be sufficient, but a comprehensive screening yields substantially more confidence.