The medicinal plant, Trachyspermum ammi is a rich source of active pharmaceutical ingredients with pharmaceutics effects. Microsatellite markers play a key role in the genome and gene expression, especially in secondary metabolite biosynthesis in medicinal plants. For the first time, transcriptome sequencing of this herb medicine was carried out to identify the microsatellite markers of this species. After pair-end sequencing with the Illumina HiSeq 2000 platform, the quality of the reads was evaluated by FastQC software, trimming was performed by Trimmomatic software and De novo assembly was done with Trinity software. In this study, 11,468 unitranscripts (7913 unigenes) were found to contain 13593 potential microsatellites. The most abundant microsatellite types were di-nucleotide (67%) and tri-nucleotide (24%). Also, six repeated SSRs were the most abundant repeats. The predominant sequence was AG / CT (31%). Sixty-five percent of SSRs were belonged to class II (10-20 nucleotides) and 35% to class I (more than 20 nucleotides). The frequency of SSRs found to be approximately one per 10.1 kB of assembled sequence. More than 57 percent of unigenes containing SSRs were blasted with carrot genome. This showed that T. ammi was an Apiaceae family member and had a high similarity to the carrot genome. A total of 3437 unigenes (43%) were categorized functionally, which among them 2,219 unigenes (64.6%) belonged to the "metabolic process" category and 71 unigenes (1.2%) were assigned to the "secondary metabolic process". In this study, 12 genes were detected in the terpenoid backbone biosynthesis pathway, that their transcripts were containing a microsatellite. These SSRs probably contribute to the genes expression and the biosynthesis of metabolites, especially secondary metabolites. The development of these markers can be used for future studies of marker-assisted selection, genetic diversity and construct genetic maps in this medicinal plant.