There is increasing medical evidence for the health benefits derived from dietary intake of carotenoid antioxidants, such as β-carotene and lutein. Enhancing the nutritional levels of vegetables would improve the nutrient intake without requiring an increase in consumption. A breeding program to improve the nutritional quality of lettuce (Lactuca sativa L.) must start with an assessment of the existing genetic variation. To assess the genetic variability in carotenoid contents, 52 genotypes including crisphead, leaf, romaine, butterhead, primitive, Latin, and stem lettuces, and wild species were planted in the field in Salinas, Calif., in the Summer and Fall of 2003 with four replications. Duplicate samples from each plot were analyzed for chlorophyll (a and b), β-carotene, and lutein concentrations by high-performance liquid chromatography (HPLC). Wild accessions (L. serriola L., L. saligna L., L. virosa L., and primitive form) had higher β-carotene and lutein concentrations than cultivated lettuces, mainly due to the lower moisture content of wild lettuces. Among major types of cultivated lettuce, carotenoid concentration followed the order of: green leaf or romaine > red leaf > butterhead > crisphead. There was significant genetic variation in carotenoid concentration within each of these lettuce types. Crisphead lettuce accumulated more lutein than β-carotene, while other lettuce types had more β-carotene than lutein. Carotenoid concentration was higher in summer than in the fall, but was not affected by the position of the plant on the raised bed. Beta-carotene and lutein concentrations were highly correlated, suggesting that their levels could be enhanced simultaneously. Beta-carotene and lutein concentrations were both highly correlated with chlorophyll a, chlorophyll b, and total chlorophyll concentrations, suggesting that carotenoid content could be selected indirectly through chlorophyll or color measurement. These results suggest that genetic improvement of carotenoid levels in lettuce is feasible.