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Thymol, as a dietary monoterpene, is a phenol derivative of cymene, which is the major component of the essential oil of Trachyspermum ammi (L.). It shows multiple biological activities: antifungal, antibacterial, antivirus and anti-inflammatory. T. ammi, commonly known as ajowan, belongs to Apiaceae and is an important medicinal seed spice. To identify the putative genes involved in thymol and other monoterpene biosynthesis, we provided transcriptomes of four inflorescence tissues of two ajowan ecotypes, containing different thymol yield. This study has detected the genes encoding enzymes for the go-between stages of the terpenoid biosynthesis pathways. A large number of unigenes, differentially expressed between four inflorescence tissues of two ajowan ecotypes, was revealed by a transcriptome analysis. Furthermore, differentially expressed unigenes encoding dehydrogenases, transcription factors, and cytochrome P450s, which might be associated with terpenoid diversity in T. ammi, were identified. The sequencing data obtained in this study formed a valuable repository of genetic information for an understanding of the formation of the main constituents of ajowan essential oil and functional analysis of thymol-specific genes. Comparative transcriptome analysis led to the development of new resources for a functional breeding of ajowan.