Although second harmonic generation (SHG) imaging has emerged as a powerful tool for imaging biological tissues with submicron resolution, the excitation wavelength dependence of SHG intensity in biological tissues is an optical property that is not fully understood so far. We first calibrate system factors which may potentially affect the accuracy of the wavelength-dependent SHG measurement. Then our calibration is validated by measuring the wavelength dependence of SHG signal from a crystal under different focusing conditions and comparing with the theoretical calculations. The good agreement between the experimental results and theoretical calculations demonstrates that we have established a reliable method to validate wavelength-dependent SHG measurement over a broad wavelength range. We also investigate the wavelength dependence of a 10-μm thick mouse tendon tissue in both forward and backward directions. It is found that SHG of mouse tendon tissue decreases monotonically for excitation from 750 to 950 nm.