High nonstructural carbohydrate (NSC) concentration in forages improves ruminant digestion and ensiling. Concentration of NSC is genetically variable and increases during the day. The objective was to evaluate the response of one cycle of divergent selection for NSC on alfalfa (Medicago sativa L.) populations harvested in the morning or the afternoon under field conditions. Two populations (NSC+ and NSC-) were obtained by intercrossing 10 genotypes selected for high or low NSC concentrations from 500 genotypes of the cultivar AC Caribou. A split plot design with time of harvest (AM: 9h00 and PM: 15h00) as main plots and populations as subplots was used. Populations were evaluated for NSC, crude protein (CP), ADF, NDF, and dry matter yield (DMY). The NSC concentration, estimated by the sum of soluble sugars and starch, was predicted using NIR spectroscopy. The NSC+ population had higher NSC (116 vs 99 g/kg DM), and lower NDF (321 vs 337 g/kg DM) and ADF (248 vs 262 g/kg DM) concentrations than the NSC- population. No difference was observed for DMY and CP concentration between populations or time of harvest. Alfalfa harvested in the afternoon had greater NSC (126 vs 87 g/kg DM) and lower NDF (313 vs 342 g/kg DM) and ADF (241 vs 268 g/kg DM) concentrations than alfalfa harvested in the morning. The increase in NSC concentration with NSC+ population or PM harvest was mainly due to an increase in starch concentration. Results indicate that there is potential to increase alfalfa NSC concentration through breeding and harvest management.