Silymarin is a flavonoid compound derived from milk thistle (Silybum marianum) seeds which has several pharmacological applications. Chalcone synthase (CHS) is a key enzyme in the biosynthesis of flavonoids; thereby, the identification of CHS encoding genes in milk thistle plant can be of great importance. In the current research, fragments of CHS genes were amplified using degenerate primers which were designed based on the conserved parts of Asteraceae CHS genes and then they were cloned and sequenced. Analysis of the resultant nucleotide and deduced amino acid sequences led to the identification of two different members of CHS gene family from milk thistle (SmCHS1 and SmCHS2). Third member full length cDNA (SmCHS3) was isolated by RACE (rapid amplification of cDNA ends), whose open reading frame contained 1239 bp including exon 1 (190 bp) and exon 2 (1049 bp) encoding 63 and 349 amino acids, respectively. In silico analysis of SmCHS3 sequence revealed that, it contains all the conserved CHS sites and shared high homology with CHS proteins from other plants. Real-time PCR analysis indicated that SmCHS1 and SmCHS3 had the highest transcript level in petals in the early flowering stage and stem of 5 upper leaves, followed by 5 upper leaves in the mid-flowering stage which are most probably involved in anthocyanin and silymarin biosynthesis.