In a method of detecting a defect, a region of a substrate may be primarily scanned using a first electron beam to detect a first defect. A remaining region of the substrate, which may be defined by excluding a portion in which the first defect may be positioned from the region of the substrate, may be secondarily scanned using a second electron beam to detect a second defect. Thus, the portion with the defect may not be scanned in a following scan process so that a scanning time may be remarkably decreased.