Abstract: In this paper, pigments had been extracted from natural brown cotton fiber(NBCF) and delinted white cottonseed hulls(DWCH) by extensive use of diagnostic reagents, UV-VIS Spectrophotometer, infrared absorption spectra and normal phase high-performance liquid chromatography (NP-HPLC) comparative analysis of their chemical compositions. The results of corresponding color reactions of two kinds of pigments showed that they were similar in main chemical constituents and all belonged to condensed tannins(CT). The dominant ingredient of both two pigments were CT with pyrocatechol structure, the main ingredient of DWCH pigments also contained resorcin structure while the content of the component with this structure was very small in NBCF pigments. The NP-HPLC method was developed to separate the two samples, the results showed that there were four compositions with one overwhelming in NBCF pigments, and various kinds of constituents were separated in DWCH pigments, however, constituents with the same molecular structure were not found in two pigments.