Abstract: To determine the content of free amino acids in flue-cured tobacco flowers, and explore their accumulation rule during the growth and development, free amino acids in three flue-cured tobacco varieties (Zhongyantexiang301, Zhongchuan208 and Yunyan87) planted in Zhucheng and Liangshan were qualitatively and quantitatively determined and the content changes during the flower growth and development were analyzed. The results showed that: (1) Nineteen free amino acids were identified in flue-cured tobacco flowers, with asparagine showing the highest content, followed by proline and both of them are the primary free amino acids in tobacco flower, accounting for over 50% of the total free amino acids. (2) The free amino acid contents in flue-cured tobacco flowers grown in Zhucheng are higher than those planted in Liangshan. The free amino acid contents of Zhongyantexiang301 are significantly higher than the other varieties. (3) The content variations in free amino acids in tobacco flowers exhibited 5 different patterns during flower development, and the variation trends of valine, isoleucine, leucine, tyrosine, lysine, histidine, arginine, asparagine, proline, and tryptophan content were consistent among different tobacco varieties. (4) The contents of free amino acids except for tryptophan are highest in the buds or flower buds, with asparagine and proline being the most abundant in the flower bud. In summary, Zhongyantexiang301 flowers planted in Zhuncheng and collected at the pre-full bloom stage exhibited the most abundant free amino acids, rendering them suitable as a flavoring agent in cigarette production.