The mature cotton (Gossypium hirsutum L.) fiber is a single cell with a typically thickened secondary cell wall. The aim of this research was to use molecular, spectroscopic and chemical techniques to investigate the possible occurrence of previously overlooked accumulation of phenolics during secondary cell wall formation in cotton fibers. Relative quantitative reverse transcription-polymerase chain reaction analysis showed that GhCAD6 and GhCAD1 were predominantly expressed among seven gene homologs, only GhCAD6 was up-regulated during secondary wall formation in cotton fibers. Phylogenic analysis revealed that GhCAD6 belonged to Class I and was proposed to have a major role in monolignol biosynthesis, and GhCAD1 belonged to Class Ⅲ and was proposed to have a compensatory mechanism for monolignol biosynthesis. Amino acid sequence comparison showed that the cofactor binding sites of GhCADs were highly conserved with high similarity and identity to bona fide cinnamyl alcohol dehydrogenases. The substrate binding site of GhCAD1 is different from GhCAD6. This difference was confirmed by the different catalytic activities observed with the enzymes. Cell wall auto-fluorescence, Fourier transform infrared spectroscopy (FTIR), high-performance liquid chromatography (HPLC) and chemical analyses confirmed that phenolic compounds were bound to the cell walls of mature cotton fibers. Our findings may suggest a potential for genetic manipulation of cotton fiber properties, which are of central importance to agricultural, cotton processing and textile industries. The aim of this research was to use molecular, spectroscopic and chemical techniques to investigate the possible occurrence of previously overlooked accumulation of phenolics during secondary cell wall formation in cotton fibers. Relative quantitative reverse transcription-polymerase chain reaction analysis showed that GhCAD6 and GhCAD1 were predominantly expressed among seven gene homologs, only GhCAD6 was up-regulated during secondary wall formation in cotton fibers. Phylogenic analysis revealed that GhCAD6 belonged to Class I and was proposed to have a major role in monolignol biosynthesis, and GhCAD1 belonged to Class III and was proposed to have a compensatory mechanism for monolignol biosynthesis. Amino acid sequence comparison showed that the cofactor binding sites of GhCADs were highly conserved with high similarity and identity to bona fide cinnamyl alcohol dehydrog This assay was previously published in French. Consultant site for GhCAD1 is different from GhCAD6. This difference was confirmed by the different catalytic activities observed with the enzymes. Cell wall auto-fluorescence, Fourier transform infrared spectroscopy (FTIR), high-performance liquid chromatography (HPLC) and chemical analyzes: that phenolic compounds were bound to the cell walls of mature cotton fibers. Our findings may suggest a potential for genetic manipulation of cotton fiber properties, which are of central importance to agricultural, cotton processing and textile industries.