Five flavonoid structural genes, encoding chalcone isomerase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, anthocyanidin synthase, and anthocyanidin reductase, were cloned from a brown-fiber cotton line (T586). The predicted proteins of these genes exhibit high sequence similarity with corresponding enzymes from various plants. RT-PCR analysis showed these genes are developmentally co-regulated and preferentially expressed in developing fibers of T586. Expression analyses and dimethylaminocinnaldehyde staining demonstrated that high transcript levels of these genes in developing fibers and presence of proanthocyanidins in mature fibers co-segregated with brown fiber in a recombination inbred line population. Our results indicated that the cloned flavonoid structural genes and proanthocyanidins were involved in the pigmentation in brown cotton fibers.