Second generation biofuels and bio-based products derived from lignocellulosic biomass are likely to replace current fuels derived from simple sugars and starch because of greater yield potential. However, the ideal biofuel cropping system would not only be capable of high aboveground dry matter yields, but also enhance or at least minimize negative effects on other ecosystem services such as soil carbon and nitrogen cycling. The objective of this research was to thus to quantify root biomass production and root decomposition rates for six perennial biofuel grasses. Giant reed [Arundo donax (L.)], elephantgrass [Pennisetum purpureum (Schum.)], energycane (Saccharum spp.), sugarcane (Saccharum spp.), sweetcane [Saccharum arundinaceum (Retz.) Jesw.], and giant miscanthus [Miscanthus × giganteus (Greef and Deuter ex Hodkinson and Renvoize)] were established in Fall 2008 near Gainesville, Florida and harvested once annually in 2008 and 2009 and twice annually in 2010 and 2011. Root decomposition rates were measured in situ over 12 months along with initial and final root tissue composition. Aboveground dry matter production was as high as 35-40 Mg/ha in 2008 and 2009, but declined on average to 20 Mg/ha in 2011 with multiple cuttings. Standing root biomass after fall harvest was higher in sweetcane (10.0 Mg ha^-1), giant miscanthus (8.98 Mg ha^-1), and giant reed (8.78 Mg ha^-1) than in sugarcane (2.50 Mg ha^-1), energycane (3.03 Mg ha^-1), and elephantgrass 3.15 (Mg ha^-1). Root potential decomposition rate constant (K_pot, g kg^-1 d^-1) was higher in elephantgrass 3.64 g kg^-1 d^-1 and sweetcane 2.77 g kg^-1 d^-1 than sugarcane 1.62 g kg^-1 d^-1 and energycane 1.48 g kg^-1 d^-1. Notably, K_pot was positively related to initial root tissue carbon, glucose, xylose, and total fiber carbohydrate concentrations, but negatively related to arabinose concentrations and specific root volume. Thus, giant reed, sugarcane, and energycane generally decomposed slowly due to root tissue composition and morphological traits. However, giant reed exhibited both high root biomass production, especially deeper in the soil profile, and slow decomposition rates favorable for soil organic matter improvement.