In a method of forming an isolation layer, first and second trenches are formed on a substrate. The first and the second trenches have first and second widths, respectively, and the second width is greater than the first width. A second isolation layer pattern partially fills the second trench. A first isolation layer pattern and the third isolation layer pattern are formed. The first isolation layer pattern fills the first trench, and the third isolation layer pattern is formed on the second isolation layer pattern and fills a remaining portion of the second trench.