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Background:Naturally-colored cotton has become increasingly popular because of their natural properties of coloration, UV protection, flame retardant, antibacterial activity and mildew resistance. But poor fiber quality and limited color choices are two key issues that have restricted the cultivation of naturally-colored cotton. To identify the possible pathways participating in fiber pigmentation in naturally-colored cottons, five colored cotton accessions in three different color types (with green, brown and white fiber) were chosen for a comprehensive analysis of phenylpropanoid metabolism during fiber development. Results:The expression levels of flavonoid biosynthesis pathway genes in brown cotton fibers were significantly higher than those in white and green cotton fibers. Total flavonoids and proanthocyanidin were higher in brown cotton fibers relative to those in white and green cotton fibers, which suggested that the flavonoid biosynthesis pathway might not participate in the pigmentation of green cotton fibers. Further expression analysis indicated that the genes encoding enzymes for the synthesis of caffeic acid derivatives, lignin and lignan were activated in the developing fibers of the green cotton at 10 and 15 days post-anthesis. Conclusions:Our results strengthen the understanding of phenylpropanoid metabolism and pigmentation in green and brown cotton fibers, and may improve the breeding of naturally-colored cottons.