A method of fabricating an isolation structure is provided. A first oxide layer and a first, second, and third hard mask layers are formed on a substrate. A patterned third hard mask layer is formed. Second oxide layers are formed on sidewalls of the patterned third hard mask layer and a fourth hard mask layer is formed between the second oxide layers. The second oxide layers and the second hard mask layer are removed using the patterned third hard mask layer and the fourth hard mask layer as a mask, to form a patterned second hard mask layer. The patterned third hard mask layer and the fourth hard mask layer are removed. A portion of the patterned second hard mask layer is removed to form trench patterns. A patterned first hard mask layer and first oxide layer, and trenches located in the substrate are defined. An isolation material is formed.