Temperately adapted perennial sorghum feedstocks have recently begun to receive increasing interest as candidate energy crops, producing significant biomass and contributing agroecological benefits including increased soil organic carbon, reduced soil erosion, reduced input requirements, and higher net energy return. Rhizomes are the primary morphological feature facilitating overwintering in Sorghum species; however, underlying physiological mechanisms governing rhizome overwintering remain poorly characterized. In this study, we investigated the composition of sorghum rhizomes from diverse germplasm before and after overwintering at two locations and three experimental environments. Significant positive correlations were found between rhizome overwintering and water-soluble carbohydrates, ethanol soluble carbohydrates, and fructan concentrations, while significant negative correlations were found between rhizome overwintering and both crude fat and starch. Near-infrared spectroscopy (NIRS) calibration equations were developed to quickly and efficiently predict the concentrations of each of these assimilates in rhizomes.