Controlled Environment Agriculture (CEA) is a method of increasing crop productivity per unit area of cultivated land by extending crop production into the vertical dimension and enabling year-round production. Light emitting diodes (LED) are frequently used as the source of light energy in CEA systems and light is commonly the limiting factor for production under CEA conditions. In the current study, the impact of different spectra was compared with the use of white LED light. The various spectra were white; white supplemented with ultraviolet b for a week before harvest; three combinations of red/blue lights (red 660 nm with blue 450 nm at 1:1 ratio; red 660 nm with blue 435 nm 1:1 ratio; red 660 nm with blue at mix of 450 nm and 435 nm 1:1 ratio); and red/blue supplemented with green and far red (B/R/G/FR, ratio: 1:1:0.07:0.64). The growth, yield, physiological and chemical profiles of two varieties of lettuce, Carmoli (red) and Locarno (green), responded differently to the various light treatments. However, white (control) appeared to perform the best overall. The B/R/G/FR promoted the growth and yield parameters in both varieties of lettuce but also increased the level of stem elongation (bolting), which impacted the quality of grown plants. There was no clear relationship between the various physiological parameters measured and final marketable yield in either variety. Various chemical traits, including vitamin C content, total phenol content, soluble sugar and total soluble solid contents responded differently to the light treatments, where each targeted chemical was promoted by a specific light spectrum. This highlights the importance of designing the light spectra in accordance with the intended outcomes. The current study has value in the field of commercial vertical farming of lettuce under CEA conditions.