Growing vegetables in controlled environments (CEs), such as hydroponics, aquaponics, and vertical structures, is a rapidly expanding industry in Florida and the United States, especially in nearby urban areas. Although lettuce (Lactuca sativa) is still mostly produced in fields, growing in CEs proximal to urban areas has become increasingly popular because it may facilitate reduced transportation time and associated postharvest degradation. Lettuce is among the top-most consumed vegetables in the United States and could provide some of the nutrition missing in the US diet. This research was planned to understand the levels of some vitamins that are key for human health, including vitamin E (tocopherols), vitamin K1 (phylloquinone), and vitamin C (ascorbic acid), in lettuce grown in greenhouse hydroponics. Lettuce germplasm was grown using the hydroponic nutrient film technique system in three greenhouse experiments: at the beginning, middle, and end of the Florida, USA, growing season (from Aug 2020 to Mar 2021). Genetic variation for these vitamins were found among the germplasm tested in the four morphological types of lettuce, romaine, Boston, Latin, and leaf. In addition, a sugar analysis was conducted in this germplasm, of which fructose was the most abundant sugar. A significant genotype × environment (G × E) interaction was observed, indicating that the levels of these compounds, especially vitamins, was environment dependent. However, the presence of certain non-crossover G × E interactions indicates that selecting lettuce in a representative environment could result in new cultivars with higher vitamin content. This research marks the initial steps to improve lettuce for these vitamins, which can contribute to better health of US consumers, not for the highest amount of these compounds in lettuce but for the offset due to its high consumption.