A multispectral camera acquires spectral color images with high fidelity by splitting the light spectrum into more than three bands. Because of the shift of focal length with wavelength, the focus of each channel should be mechanically adjusted in order to obtain sharp images. Because progressive adjustment is quite time consuming, the clear focus must be determined by using a limited number of images. This paper exploits the symmetry of focus measure distribution and proposes a simple yet efficient autofocus method. The focus measures are computed using first-order image derivatives, and the focus curve is obtained by spline interpolation. The optimal focus position, which maximizes the symmetry of the focus measure distribution, is then computed according to distance metrics. The effectiveness of the proposed method is validated in the multispectral camera system, and it is also applicable to relevant imaging systems.