A fully automated method for identification and quantification of five polar pesticides in groundwater by isotope dilution-online solid-phase extraction (SPE) coupled with high-performance liquid chromatography-quadrupole Orbitrap high-resolution mass spectrometry was developed. After one step of filtration, an aliquot of a 7.5-ml water sample was automatedly preconcentrated and purified on a turbulent Cyclone SPE column. The analytes were eluted in backflush mode, then separated on an analytical column and acquired by full MS/dd-MS 2 scan in negative and positive ions mode.The major parameters for sample loading, cleanup, and elution were optimized in detail. Preconcentration and ionization efficiency were highly improved by using 0.1% acid solution in the mobile phase. The method provided good linearity of calibration coefficients (R 2 > 0.995), sensitive method limits of detection (0.5-10.0 ng/L), accurate mass spectra (within 5 ppm error), satisfactory matrix spiking recoveries (98.4% to 109%), and high precision (intraday/interday relative standard deviations 1.57-8.90%). The method was successfully applied to analyze large batch groundwater of National Groundwater Monitoring Project and suspect screening of potential pesticides in groundwater. The study provided a practical alternative for a simple, robust, sensitive, and accurate identification and qualification of five polar pesticides in groundwater.