Three field experiments of nitrogen (N) rates, plant growth regulator (PIX) applications, and irrigation regimes were conducted in 2001 and 2002 to investigate relationships between hyperspectral reflectance (400-2500 nm) and cotton (Gossypium hirsutum L.) growth, physiology, and yield. Leaf and canopy spectral reflectance and leaf N concentration were measured weekly or biweekly during the growing season. Plant height, mainstem nodes, leaf area, and aboveground biomass were also determined by harvesting 1-m row plants in each plot at different growth stages. Cotton seed and lint yields were obtained by mechanical harvest. From canopy hyperspectral reflectance data, several reflectance indices, including simple ratio (SR) and normalized difference vegetation index (NDVI), were calculated. Linear relationships were found between leaf N concentration and a ratio of leaf reflectance at wavelengths 517 and 413 nm (R517/R413) (r2 = 0.70, n = 150). Nitrogen deficiency significantly increased leaf and canopy reflectance in the visible range. Plant height and mainstem nodes were related closely to a SR (R750/R550) according to either a logarithmic or linear function (r2 = 0.63~0.68). The relationships between LAI or biomass and canopy reflectance could be expressed in an exponential fashion with the SR or NDVI [(R935-R661)/(R935+R661)] (r2 = 0.67~0.78). Lint yields were highly correlated with the NDVI around the first flower stage (r2 = 0.64). Therefore, leaf reflectance ratio of R517/R413 may be used to estimate leaf N concentration. The NDVI around first flower stage may provide a useful tool to predict lint yield in cotton.