In a method of manufacturing a semiconductor device, an isolation pattern is formed on a substrate. The isolation pattern includes an opening that exposes a portion of the substrate. A preliminary polysilicon layer is formed on the substrate and the isolation pattern to partially fill up the opening. A sacrificial layer is formed on the preliminary polysilicon layer. The sacrificial layer is partially etched to expose a portion of the preliminary polysilicon layer formed on a shoulder portion of the isolation pattern. A first polysilicon layer is formed by etching the exposed portion of the preliminary polysilicon layer to enlarge an upper width of the opening. After the etched sacrificial layer is removed, a second polysilicon layer is formed on the first polysilicon layer to fill up the enlarged opening. Because the upper width of the opening is larger than the lower width, no seam or void would be generated in the second polysilicon layer, therefore improving the electrical characteristics and reliability of the device.