The concentration of stem cell wall constituents in alfalfa, Medicago sativa L., herbage can affect dry matter intake and energy availability in dairy and beef production systems, and impact energy conversion efficiency when alfalfa is used to produce biofuels. Stem Klason lignin, glucose, xylose, and uronic acid and total cell wall concentrations are effected by maturity and environmental influences. Knowledge of the environmental influence and variability of the stem cell wall component traits will be essential in a plant breeding program with goals to improve these traits. We initiated a study using near infrared reflectance spectroscopy (NIRS) calibrated by wet chemistry to identify individual alfalfa plants that differed (highest and lowest) in stem Klason lignin, glucose, xylose, and uronic acid and total cell wall concentrations. Individual plants that differed in these stem traits were vegetatively propagated and clones of each selected plant were established in replicated experiments at two field locations. All clones were sampled at 25 % bloom for spring, early, and late summer harvests for two years and evaluated using NIRS calibration equations for each of these stem cell wall component traits. Results indicated that alfalfa stem glucose, uronic acid, and Klason lignin concentrations demonstrated repeatability and environmental stability suggesting potential for improvement for these stem cell wall component traits.