Abstract. Non-mydriatic retinal imaging is an important tool for diagnosis and progression assessment of ophthalmic diseases. Because it does not require pharmacological dilation of the patient's pupil, it is essential for screening programs performed by non-medical personnel. A typical camera is equipped with a manual focusing mechanism to compensate for the refractive errors in the eye. However, manual focusing is error prone, especially when performed by inexperienced photographers. In this work, we propose a new and robust focus measure based on a calculation of image anisotropy which, in turn, is evaluated from the directional variance of the normalized discrete cosine transform. Simulation and experimental results demonstrate the effectiveness of the proposed focus measure.