This study evaluated the effect of variety (V; CDC Cowboy, CDC Copeland, and Xena) and stage of harvest maturity (M; milk, early-, mid-, and hard-dough) on nutrient and neutral detergent fiber digestibility (NDFD) characteristics of barley forage using a randomized complete block design with 3 × 4 factorial treatment arrangement. Barley varieties had similar crude protein, but CDC Cowboy had greater (P < 0.01) acid detergent fiber (ADF), neutral detergent fiber (NDF), and lignin, and lower (P < 0.01) total digestible nutrient (TDN) content relative to Xena. Starch content of CDC Cowboy was lower (P < 0.01) than Xena at all stages of maturity with CDC Copeland intermediate at early- and mid-dough stages. Crude protein, ADF, NDF, and lignin content decreased (P < 0.01) while starch and TDN content increased (P < 0.01) with advancing maturity. Xena had greater (P < 0.01) NDFD, as determined by 6 h in vitro incubation using Daisy^II system (NDFD_6h), at milk, mid- and hard-dough stages of maturity than CDC Cowboy with CDC Copeland intermediate at mid-dough. However, CDC Cowboy had greater (P < 0.01) NDFD, as determined by 30 h in vitro incubation using Daisy^II system (NDFD_30h), at early-dough stage than Xena and greater NDFD_30h at hard-dough stage than CDC Copeland. Xena had the lowest (P < 0.01) indigestible NDF based on 288 h ruminal in situ incubation (INDF_288h), relative to CDC Copeland. These results indicate that to optimize NDFD_30h, variety should be considered when deciding the timing of harvest.