Abstract Neonicotinoids are systemic insecticides used since the 1990’s , that possess renal tubular toxicity. We conducted a field-based descriptive study in the North Central Dry-zone of Sri Lanka, where chronic kidney disease (CKD) of unknown etiology has been increasing since the 1990’s. To elucidate the relationship between renal tubular dysfunctions and urinary neonicotinoids concentrations, we collected spot urine samples from15 CKD patients, 15 family members, and 62 neighbors in 2015, analyzed two renal tubular biomarkers, Cystatin-C and L-FABP, quantified seven neonicotinoids and a metabolite N -desmethyl-acetamiprid by LC–MS/MS; and we investigated their symptoms using a questionnaire. Cystatin-C and L-FABP had a positive correlation ( p < 0.001). N -Desmethyl-acetamiprid was detected in 92.4% of the urine samples, followed by dinotefuran (17.4%), thiamethoxam (17.4%), clothianidin (9.8%), thiacloprid and imidacloprid. Dinotefuran and thiacloprid have never been registered in Sri Lanka. In High Cystatin-C group (> 70 μg/gCre, n = 7), higher urinary concentration of dinotefuran ( p = 0.009), and in Zero Cystatin-C group (< LOQ, n = 7), higher N -desmethyl-acetamiprid ( p = 0.013), dinotefuran ( p = 0.049), and thiacloprid ( p = 0.035), and more complaints of chest pains, stomachache, skin eruption and diarrhea ( p < 0.05) were found than in Normal Cystatin-C group (n = 78). Urinary neonicotinoids may be one of the potential risk factors for renal tubular dysfunction in this area.