Systems and methods are disclosed for focusing a microscope using images acquired under multiple illumination conditions. In one implementation, an autofocus microscope may include an image capture device, a focus actuator, an illumination assembly, and a controller. The controller may cause the illumination assembly to illuminate a sample at a first illumination condition and at a second illumination condition. The controller may acquire a first image of the sample illuminated from the first illumination angle and a second image of the sample illuminated from the second illumination angle. The controller may further determine an amount of shift between image features present in the first image of the sample and a corresponding image features present in the second image of the sample, If the amount of determined shift is non-zero, the focus actuator may change the distance between the sample and the focal plane.