Methods of manufacturing a semiconductor device, which can reduce hot electron induced punchthrough (HEIP) and/or improve the operating characteristics of the device include selectively forming an oxynitride layer in a device isolation layer according to the characteristics of transistors isolated by the device isolation layer. The methods include forming first trenches and second trenches on a substrate, forming an oxide layer on the surfaces of the first trenches and the second trenches, selectively forming an oxynitride layer on the second trenches by using plasma ion immersion implantation (PIII), and forming a buried insulating layer in the first trenches and the second trenches. The buried insulating layer may be planarized to form a first device isolation layer in the first trenches and a second device isolation layer in the second trenches.