Napier grass (Pennisetum purpureum Schum.) is one of the highest-yielding feedstocks for bio-based products and biofuel in semi-tropical areas of the USA and the world. Thirty genetically diverse Napier grass accessions were selected from a germplasm nursery in Tifton, GA and analyzed for fiber, ash, nitrogen (N) concentration, and biochemical conversion to ethanol. A near-infrared reflectance spectroscopy (NIRS) calibration was developed from this material to predict ethanol production, xylans, N concentration, and ash by separating leaves and stems and correlating with wet chemistry analyses. The high diversity of material from dwarf material with high leaf and stem digestibility to taller and more productive Napier grass cultivars resulted in high correlations with predicted results for in vitro dry matter digestibility (2 = 0.93), neutral detergent fiber (r2 = 0.83), acid detergent fiber (r2 = 0.95), ethanol (r2 = 0.90), nitrogen (r2 = 0.99), and ash (r2 = 0.98). This information will allow faster evaluation of Napier grass biomass for use by industry or geneticists.