In a method of detecting a defect of a substrate, a first light having a first intensity may be irradiated to a first region of the substrate through a first aperture. A defect in the first region may be detected using a first reflected light from the first region. A second light having a second intensity may be irradiated to a second region of the substrate through a second aperture. A defect in the second region may be detected using a second reflected light from the second region. Thus, the defects by the regions of the substrate may be accurately detected.