Neonicotinoids have been previously detected in Iowa surface waters, but less is known regarding their occurrence in groundwater. To help fill this research gap, a groundwater study was conducted in eastern Iowa and southeastern Minnesota, a corn and soybean producing area with known heavy neonicotinoid use. Neonicotinoids were studied in alluvial aquifers, a hydrogeologic setting known to be vulnerable to surface-applied contaminants. Groundwater samples were analyzed from 40 wells for six neonicotinoid compounds (acetamiprid, clothianidin, dinotefuran, imidacloprid, thiacloprid, thiamethoxam), and sulfoxaflor. Samples were analyzed using liquid chromatography tandem mass spectrometry (LC/MS/MS) with both direct aqueous injection and solid phase extraction methods. Neonicotinoids were prevalent in the alluvial aquifers with 73% of the wells having at least one neonicotinoid detection. Clothianidin (68%, max: 391.7 ng/L) was the most commonly detected, followed by imidacloprid (43%, max: 6.7 ng/L) and thiamethoxam (3%, max: 0.2 ng/L). Acetamiprid, dinotefuran, sulfoxaflor, and thiacloprid were not detected during the study. The solid phase extraction method was more sensitive than direct aqueous injection, where only clothianidin detected in 23% of samples. SPE is the preferred method for detecting low concentrations of hydrophilic pesticides in water. This study documented that the combination of heavy chemical use overlying a hydrogeologic setting vulnerable to surface applied contaminants leads to transport of neonicotinoids into an important groundwater resource.