Saturejais rich in phenolic monoterpenoids, mainly carvacrol, that is of interest due to diverse biological activities including antifungal and antibacterial. However, limited information is available regarding the molecular mechanisms underlying carvacrol biosynthesis and its regulation for this wonderful medicinal herb. To identify the putative genes involved in carvacrol and other monoterpene biosynthesis pathway, we generated a reference transcriptome in two endemicSaturejaspecies of Iran, containing different yields (Satureja khuzistanicaandSatureja rechingeri). Cross-species differential expression analysis was conducted between two species ofSatureja. 210 and 186 transcripts related to terpenoid backbone biosynthesis were identified forS.khuzistanicaandS.rechingeri, respectively. 29 differentially expressed genes (DEGs) involved in terpenoid biosynthesis were identified, and these DEGs were significantly enriched in monoterpenoid biosynthesis, diterpenoid biosynthesis, sesquiterpenoid and triterpenoid biosynthesis, carotenoid biosynthesis and ubiquinone and other terpenoid-quinone biosynthesis pathways. Expression patterns ofS.khuzistanicaandS.rechingeritranscripts involved in the terpenoid biosynthetic pathway were evaluated. In addition, we identified 19 differentially expressed transcription factors (such as MYC4, bHLH, and ARF18) that may control terpenoid biosynthesis. We confirmed the altered expression levels of DEGs that encode carvacrol biosynthetic enzymes using quantitative real-time PCR (qRT-PCR). This study is the first report onde novoassembly and transcriptome data analysis inSaturejawhich could be useful for an understanding of the main constituents ofSaturejaessential oil and future research in this genus.