High-resolution measurements obtained from NOAA "best" atmospheric turbulence (BAT) probes mounted on an EGRETT high-altitude research aircraft were used to characterize turbulence in the upper troposphere and lower stratosphere at scales from 2 m to 20 km, focusing on three-dimensional behavior in the sub-kilometer-scale range. Data were analyzed for 129 separate level flight segments representing 41 h of flight time and 12 600 kmof wind-relative flight distances. The majority of flights occurred near the tropopause layer of the winter subtropical jet stream in the Southern Hemisphere. Second-order structure functions for velocity and temperature were analyzed for the separate level-flight segments, individually and in various ensembles.A3Dscaling range was observed at scales less than about 100 m, with power-law exponents for the structure functions of the velocity component in the flight direction varying mostly between 0.4 and 0.75 for the separate flight segments, but close to 2/3 for the ensemble-averaged curves for all levels and for various subensembles. Structure functions in the3Dscaling range were decoupled from those at scales greater than 10 km, with the large-scale structure functions showing less variation than those at smaller scales. Weakly anisotropic behavior was observed in the 3D range, with structure parameters for the lateral and vertical velocities on the same order as those in the flight direction but deviating from the expected isotropic value. Anisotropy was correlated with turbulence intensity, with greater anisotropy associated with weaker turbulence.