To investigate the potential for the utilization of cotton stalk, ammonia hydrothermal treatment was applied to fractionate the samples into aqueous ammonia-soluble and ammonia-insoluble portions. The ammonia-soluble portion was purified to yield lignin fractions. The lignin fractions obtained were characterized by wet chemistry (carbohydrate analysis) and spectroscopy methods (FT-IR, C-13 and H-1-C-13 HSQC NMR spectroscopy) as well as gel permeation chromatography (GPC). The results showed that the cotton stalk lignin fractions were almost absent of neutral sugars (0.43%-1.29%) and had relatively low average molecular weights (1255-1746 g/mol). The lignin fractions belonged to typical G-S lignin, which was composed predominately of G-type units (59%) and noticeable amounts of S-type units (40%) together with a small amount of H-type units (similar to 1%). Furthermore, the ammonia-extractable lignin fractions were mainly composed of beta-O-4' inter-unit linkages (75.6%), and small quantities of beta-beta' (12.2%), together with lower amounts of beta-5' carbon-carbon linkages (7.4%) and p-hydroxycinnamyl alcohol end groups.