Brown cotton is a kind of naturally colored cotton which accumulates brown pigment in fiber. In the experiment, the variations of DNA methylation and genes expression were studied during the development processes from 5 DPA to 25 DPA for brown fiber. By using the methylation-sensitive amplified polymorphism technique, we choose 66 pairs of selective-amplification primers to assess the status and levels of cytosine methylation. The hemimethylation of the external cytosine and the full methylation of the internal cytosine were scored. As a result, with the development of fiber from 5 DPA to 25 DPA, the ratios of the external hemimethylation and internal full methylation were gradually increased. In detail, at the stages of 5, 10, 15 and 25 DPA, the percentages of external hemimethylation were 8.29, 8.81, 9.77 and 10.09 %, the ratios of internal full methylation were 17.91, 19.20, 20.02 and 20.78 %, respectively. The development of brown fiber triggered the increase of methylated loci in the whole genome. For further analysis, we used cDNA-AFLP protocol to identify the specially expressed genes for further analysis. Totally, 30 polymorphic transcription-derived fragments (TDFs) were isolated and sequenced. By homologous alignment analysis, 19 TDFs were found similar with genes from cotton. Among them, the homologous genes of TDF5, TDF6, TDF10, TDF12, TDF17, TDF22, TDF23 and TDF25 were all from fiber. Furthermore, 13 TDFs were found to be homologous with reported functional genes.