Chlorine is a disinfectant commonly used to treat water. The United States Environmental Protection Agency (USEPA) has set a standard limit of up to 4 mg·L−1 chlorine for drinking water. The objective of this project was to identify chlorine phytotoxicity thresholds on ‘Rex’ lettuce (Lactuca sativa) when the water source contained chlorine levels within the USEPA standard limits. The nutrient solution to grow lettuce was prepared with reverse osmosis–treated water treated with 0, 0.5, 1, 1.5, 2, and 4 mg·L−1 chlorine and then fertilizers were added. Lettuce plants were grown in a deep-water culture hydroponic system. Visual toxicity symptoms on leaves, relative leaf greenness, and fresh and dry biomass were measured. Our results indicate that irrigation water sources with ≥1 mg·L−1 chlorine used to prepare nutrient solutions can cause phytotoxicity in lettuce plants in just 3 days. Compared with the untreated control, lettuce shoot biomass was lower by 30%, 55%, 66%, 83%, and 92% at 0.5, 1, 1.5, 2, and 4 mg·L−1 of chlorine, respectively. Water sources with ≥ 1 mg·L−1 chlorine can cause significant marketable yield reduction in lettuce grown in deep-water culture.