This work reports on systematic investigation of the structure and thermal conductivity of lignin-based carbon fibers (CF) at the microscale. The lignin-based CF is produced by melt-spinning pyrolytic lignin derived from red oak. The 0 K-limit phonon scattering mean free path uncovers a characteristic structure size of ∼1.2 nm, which agrees well with the crystallite size by X-ray scattering (0.9 and 1.3 nm) and the cluster size by Raman spectroscopy (2.31 nm). The thermal conductivity of as-prepared CFs is determined at ∼1.83 W/m·K at room temperature. The thermal reffusivity of CFs shows little change from room temperature down to 10 K, uncovering the existence of extensive defects and grain boundaries which dominate phonon scattering. The localized thermal conductivity of CFs is increased by more than ten-fold after being annealed at ∼2800 K, to a level of 24 W/m·K. Our microscale Raman scanning from less annealed to highly annealed regions shows one-fold increase of the cluster size: from 1.83 nm to 4 nm. This directly confirms structure improvement by annealing. The inverse of the thermal conductivity is found linearly proportional to the annealing temperature in the range of 1000–2800 K.