A semiconductor device having multi-gate insulating layers and methods of fabricating the same are provided. The semiconductor device includes an isolation region disposed at a predetermined region of a semiconductor substrate. The isolation region defines at least one first active region and at least one second active region. The first active region is covered with a first gate insulating layer, and the second active region is covered with a second gate insulating layer which is thinner than the first gate insulating layer. Preferably, the top surface of the first gate insulating layer has the same height as the that of the second gate insulating layer. The isolation region is filled with an isolation layer which preferably covers the entire sidewalls of the first and second gate insulating layers. A typical method includes the step of selectively forming a first gate insulating layer at a predetermined region of a semiconductor substrate. A second gate insulating layer which is thinner than the first insulating layer is selectively formed at the surface of the semiconductor substrate adjacent to the first gate insulating layer. Preferably, the bottom surface of the first gate insulating layer is lower than that of the second gate insulating layer. The first and second gate insulating layers are covered with a conductive layer. The conductive layer, the first and second gate insulating layers, and the substrate are etched to form an isolation region, for example, a trench region, defining a first active region under the first gate insulating layer and a second active region under the second gate insulating. An isolation layer is formed in the trench region. The isolation layer preferably covers the entire sidewalls of the first and second gate insulating layers.