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Graphical abstract.This study aimed to use micro-FTIR with transmission mode to investigate cellulose crystallinity of developing cotton fibers. Compared with ATR-FTIR method, we found that micro-FTIR can obtain more information of cellulose inside of the developing cotton fibers, especially in high wavenumber of 2800-3000 cm-1 region. Combined with curve fitting method, a new IR crystallinity index (CI) method named wax crystallinity index (WCI) was introduced to evaluate the cellulose crystallinity in the development of cotton fibers