Hemp seed oil, known for its abundance in polyunsaturated fatty acids, is considered an emerging nutritious alternative to conventional edible oils available in the market. However, the knowledge about the oil quality and stability during cooking is limited. The impact of different cooking temperatures (170 °C, 190 °C, 210 °C, 230 °C) and times (15 min, 30 min, 1 h, 2 h, 3 h) on the fatty acid profiles and physicochemical properties (peroxide and acid values) of three types of hemp seed oil (original hemp oil, garlic infused hemp oil, chili infused hemp oil) were investigated in this study. When subjected to the tested heating temperatures and times, the peroxide and acid values of these oils remained within acceptable limits stated in the Codex standard. The two infused hemp seed oils were found to have better oxidative stability than the original ones. GC-FID analysis indicated that linoleic acid was the predominant fatty acid, followed by γ-linolenic acid and cis-9 oleic acid. Two trans fatty acids (trans-9 elaidic acid, linolelaidic acid) were detected at low levels in the oils, and no generation of new trans fatty acids was observed following the heating process. To ensure the quality and safety of hemp seed oils, it is recommended to limit heating treatment below 190 °C for durations of less than 1 h. This study provided practical information on the safe usage of hemp seed oil in cooking and served as a foundation for further research.