The authenticity of food products marketed as health-promoting foods—especially unrefined, cold-pressed seed oils—should be controlled to ensure their quality and safeguard consumers and patients. Metabolomic profiling using liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC–QTOF) was employed to identify authenticity markers for five types of unrefined, cold-pressed seed oils: black seed oil (Nigella sativaL.), pumpkin seed oil (Cucurbita pepoL.), evening primrose oil (Oenothera biennisL.), hemp oil (Cannabis sativaL.) and milk thistle oil (Silybum marianum). Of the 36 oil-specific markers detected, 10 were established for black seed oil, 8 for evening primrose seed oil, 7 for hemp seed oil, 4 for milk thistle seed oil and 7 for pumpkin seed oil. In addition, the influence of matrix variability on the oil-specific metabolic markers was examined by studying binary oil mixtures containing varying volume percentages of each tested oil and each of three potential adulterants: sunflower, rapeseed and sesame oil. The presence of oil-specific markers was confirmed in 7 commercial oil mix products. The identified 36 oil-specific metabolic markers proved useful for confirming the authenticity of the five target seed oils. The ability to detect adulterations of these oils with sunflower, rapeseed and sesame oil was demonstrated.