To isolate and characterize chalcone synthase gene in anthocyanin biosynthetic pathway during flower development of Dendrobium Sonia Earsakul. The gene was isolated from floral tissues of the orchid by reverse transcriptase polymerase chain reaction. Characterization of the gene considered to its relatedness to chalcone synthase gene in other orchid plants elucidated by construction of a neighbor-joining phylogenetic tree. Gene expression pattern related to flower development and pigmentation was investigated by relative quantification real time polymerase chain reaction. A complete coding sequence was obtained and sequence analysis revealed that the gene of Dendrobium Sonia Earsakul consisted of 1,188 bp. Blast analysis and multiple alignments showed that the chalcone synthase gene of Dendrobium Sonia Earsakul shares high homology to chalcone synthase gene of Dendrobium genus particularly Dendrobium hybrid Uniwai prince. Phylogenetic tree revealed that chalcone synthase of Dendrobium genus are highly conserved. The chalcone synthase gene of Dendrobium Sonia Earsakul was highly expressed in young flower bud with no pigmentation and the expression was sharply decreased when young flower bud started accumulation of pigments. Expression of chalcone synthase gene was then maintained at the same level until young bud developed into fully opened flowers.