Root systems in an American sycamore (Platanus occidentalis L.) biomass plantation were sampled to estimate total biomass and C and N content to a soil depth of 120 cm. Trees were grown at a 1.5- x 3.0-m spacing without irrigation on loessal silt loam soil of a former soybean field in southwestern Tennessee, USA. Root systems of 10 six-year-old trees (diameter = 7.7-10.2 cm) were sampled using a two-phase approach. In phase I, roots were sampled by taking soil core samples (n=150) from the areas between trees and subsequently separating roots from soil by washing. In phase II, the central root system of each tree, which consisted of the larger roots near the stem, was extracted from the ground with a backhoe. Extracted roots were cleaned and total C and N concentrations were determined by combustion analysis. Data from phase I were used to estimate root mass in 5.0-m3 of soil between trees, while phase II determined root mass in 0.4-m3 of soil surrounding the trees' central root systems. Phase I and II data were combined to estimate total biomass, C, and N content of root systems to a soil depth of 120 cm. Estimated dry biomass of roots per tree averaged 6.21 kg, 5.35 kg in the central root system (phase II) and 0.86 kg in the outer root system (phase I). Estimated total dry biomass of root systems was 13.4 Mg ha-1. Estimated C and N content of root systems was 6.4 Mg ha-1 and 112 kg ha-1, respectively. While many strategies exist for estimating root system biomass, a multi-phase approach to sampling is an efficient technique in woody biomass plantations.