The purpose of this study is to objectively investigate image sharpness of the metaphase chromosomes at different scanning speeds. In cytogenetic imaging, high scanning speed is sometimes applied to optimize the efficiency. However, at high scanning speed, the obtained image may be deteriorated, due to the scanning blur, under exposure and random vibration of the scanning stage. In this study, the image sharpness of metaphase chromosomes is objectively evaluated. A standard resolution target and several metaphase chromosomes are first imaged under different speeds, by a prototype scanning microscope. Then the sharpness of the acquired images is objectively assessed using a sharpness function. For this prototype system, the results demonstrate that the image sharpness of the metaphase chromosomes is optimized when the scanning speed is 0.8mm/s, under the specified experimental conditions. The results of this study may be useful for optimizing the efficiency and quality of clinical cytogenetic imaging procedures.