The polysaccharide LbGp1 from Lycium barbarum L. was sulphated with sulphur trioxide–pyridine complex in DMF, yielding two sulphated polysaccharides, which were LbGp1-OL-SL with 13.7% sulphate content, and LbGp1-OL-SH with 27.4% sulphate content. The sulphation patterns were analysed using a GC–MS strategy. After a series of sequential chemical derivatisations, the sulphated polysaccharides were converted to partially methylated alditol acetates. All the sulphate groups were replaced by acetyl groups, maintaining the positional information of the original sulphation pattern. The number and position of sulphate substitutions were deduced by comparing the relative molar ratio from methylation analysis between native polysaccharide and sulphated derivatives. In LbGp1-OL-SL, 12.65% of sulphation located on C-5 of Ara, only 0.69% and 0.34% of sulphation occurred on C-4 and C-6 Gal, respectively; while in LbGp1-OL-SH, 24.96% of sulphate groups were found at C-5 of Ara, and 0.40% and 2.02% of sulphate groups were found at C-4 and C-6 Gal, respectively.