The method of manufacturing the semiconductor device includes forming a trench to be an alignment mark in a semiconductor substrate, forming a mask film exposing a region to be a device isolation region and covering a region to be a device region by aligning with the alignment mark above the semiconductor substrate with the trench formed in, anisotropically etching the semiconductor substrate with the mask film as a mask to form a device isolation trench in the region to be the device isolation region of the semiconductor substrate, and burying the device isolation trench by an insulating film to form a device isolation insulating film. In forming the trench, the trench is formed in a depth which is smaller than a depth equivalent to a thickness of the mask film.