To clone genes involved in the betalain biosynthesis pathway and to assess the effects of phytohormones on betalain biosynthesis in Amaranthus tricolor. Five betalain biosynthesis genes were cloned by reverse transcription PCR and rapid amplification of cDNA ends. Betacyanin analyses revealed that pigments accumulated differently in various tissues and under different phytohormone treatments. Quantitative RT-PCR analysis indicated that gene expression levels did not correlate with pigment accumulation. Notably, gene expression and pigment accumulation were negatively regulated by 2,4-dichlorophenoxyacetic acid. The expression of AmaDOPA5-GT, AmaDODA, and AmaB5-GT was induced by pigmentation-promoting 6-benzyl aminopurine (6-BA). and pigmentation-inhibiting gibberellin A(3) while AmaTyDC expression was suppressed. AmaTyDC expression was also suppressed by pigmentation-promoting kinetin. Additionally, the expression of AmaB6-GT was suppressed by 6-BA. The changes in betacyanin levels among various tissues and following phytohormone treatments were related to the differences in betalain biosynthesis gene expression levels.