Black cumin, Nigella sativa L. belonging to Ranunculaceae family, is a valuable medicinal plant due to the taste and curative effects of seed and oil. Although there are studies on the agronomical and chemical characterization of black cumin, there is not any study on the thymoquinone yield, and the thymoquinone content in the cold pressed seed oil. The study investigated thymoquinone content in cold-pressed seed oil and the thymoquinone yield per hectare of Turkish black cumin genotypes from two cultivated Nigella species, N. sativa and N. damascena, along with their agronomic properties, seed yield, and fatty acid composition. Field studies were carried out in the same climatic and soil conditions in a randomized block design for two years. The fatty acid composition of the seed oils was analyzed by gas chromatography flame ionization detector (GC-FID), and the amount of thymoquinone in the cold-pressed seed oil was analyzed by the Waters Acquity UPC2 system. As a result of the field and analytic studies, significant differences were observed in other agronomic characteristics, except for the number of branches. In the studies, the highest seed yield was obtained in the Genotype 3 with 2.690 kg/ha, Genotype 17 with 2.227 kg/ha and Genotype 16 with 2.003 kg/ha. However, the variations were observed in the yields under the changing climatic conditions of the years. In the study, the seed oil ratios of the genotypes varied between 33.8% and 38.4% in the first year, and between 28.8% and 38.7% in the second year, but the difference between the genotypes in the second year was statistically significant (p < 0.05). While thymoquinone was not found in the damascena seed oil samples, it was found between 0.45% and 4.57% in the cold-pressed seed oil of the sativa genotypes. Thymoquinone yields per hectare were between 1.24 and 18.41 kg/ ha. Linoleic, oleic, and palmitic acids are the major fatty acids, and their values were found close to each other in the all genotypes. As a result of the study, the genotypes with high thymoquinone content were classified as medicine and folk medicine, and the genotypes with low thymoquinone content were classified as spice and food. It was also concluded that the sectoral classification, called bitter black cumin or sweet black cumin, is related to the thymoquinone contents in the cold-pressed seed oil.