Semiconductor devices including a trench isolation layer are provided. The semiconductor device includes a substrate having a trench therein, a liner insulation layer that covers a bottom surface and sidewalls of the trench and includes micro trenches located at bottom inner corners of the liner insulation layer, a first isolating insulation layer filling the micro trenches and a lower region of the trench that are surrounded by the liner insulation layer, and a second isolating insulation layer filling the trench on the first isolating insulation layer. The liner insulation layer on sidewalls of an upper region of the trench having a thickness that gradually increases toward a bottom surface of the trench, and the liner insulation layer on sidewalls of the lower region of the trench having a thickness that is uniform. Related methods are also provided.