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 and less competition with food production. Besides the high aboveground biomass production, these bioenergy grasses also exhibit extensive root systems. The decomposition of root biomass greatly influences nutrient cycling and microbial activity and subsequent accumulation of carbon (C) in the soil. The objective of this research was thus to characterize root morphological and chemical differences in six perennial grass species in order to better understand root decomposition and belowground C cycling of these bioenergy cropping systems. Giant reed (Arundo donax), elephantgrass (Pennisetum purpureum), energycane (Saccharum spp.), sugarcane (Saccharum spp.), sweetcane (Saccharum arundinaceum), and giant miscanthus (Miscanthus × giganteus) were established in Fall 2008 in research plots near Gainesville, Florida. Root decomposition rates were measured in situ from root decomposition bags over 12 months along with initial and final root tissue composition. Root potential decomposition rate constant (K) was higher in elephantgrass (3.64 g kg−1 day−1) and sweetcane (2.77 g kg−1 day−1) than in sugarcane (1.62 g kg−1 day−1) and energycane (1.48 g kg−1 day−1). Notably, K was positively related to initial root tissue total C (Total C), total fiber glucose (TFG), total fiber xylose (TFX), and total fiber carbohydrate (TFC) concentrations, but negatively related to total fiber arabinose (TFA) and lignin (TL) concentrations and specific root volume (SRV). Among the six species, elephantgrass exhibited root traits most favorable for fast decomposition: high TFG, high TFX, high TFC, high specific root length (SRL), and a low SRV, whereas giant reed, sugarcane, and energycane exhibited slow decomposition rates and the corresponding root traits. Thus, despite similar aboveground biomass yields in many cases, these species are likely to differentially affect soil C accumulation.