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 based on the conserved parts of Asteraceae 𝐶𝐻𝑆 genes, and then 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, 𝑆𝑚𝐶𝐻𝑆1 and 𝑆𝑚𝐶𝐻𝑆2. Third member, full-length cDNA (𝑆𝑚𝐶𝐻𝑆3) was isolated by rapid amplification of cDNA ends (RACE), 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 contains all the conserved CHS sites and shares 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 in the stem of five upper leaves, followed by five upper leaves in the mid-flowering stage which are most probably involved in anthocyanin and silymarin biosynthesis.