Wall shear stress (WSS) is of fundamental physiological and pathological significance. Current measurement technologies suffer from poor spatial resolution, or cannot measure instantaneous values in a label-free manner. Here we demonstrate dual-wavelength third-harmonic-generation (THG) line-scanning imaging, for instantaneous wall shear rate and WSS measurement in vivo. We used soliton self-frequency shift to generate dual-wavelength femtosecond pulses. Simultaneous acquisition of dual-wavelength THG line-scanning signals extract blood flow velocities at adjacent radial positions for instantaneous wall shear...