Carboxymethyl cellulose was synthesized in two-phase medium containing benzene, ethanol, and water. The distribution of substitution of hydroxyl group attached to C-k Carbons (k = 2, 3, and 6) in an anhydroglucose unit (AGU) was determined by H-1-NMR method. It showed that the sequence of distribution of carboxymethyl group of the products in AGU was C-6 > C-2 > C-3; and when degree of substitution (DS) was low to 1.0, the approximate value of C-2:C-3:C-6 was 1.45:1:2.15. When DS was higher than 1.0 and increased, the distribution of substitution tends to identical. Through analysis of X-ray diffraction and calculation of crystallinity and interplanar distance of CMC, the principle of distribution of the substituent had been explained. The influence of benzene on distribution of substituent of CMC was also investigated. It was found that distribution of substituent of CMC prepared in medium benzene-ethanol at C-6 position was higher than that prepared in ethanol.