N,N,N-Trimethylated chitosan (TMC) with varying degree of quaternization (DQ) is currently being investigated in mucosal drug, vaccine and in gene delivery. However, besides N-methylation, O-methylation and chain scission occur during the synthesis of this polymer. Since both side reactions may affect the polymer characteristics, there is a need for TMCs without O-methylation and disparities in chain lengths while varying the DQ. In this study, O-methyl free TMC with varying DQs was successfully synthesized by using a two-step method. First, chitosan was quantitatively dimethylated using formic acid and formaldehyde. Then, in the presence of an excess amount of iodomethane, TMC was obtained with different DQs by varying reaction time. TMC obtained by this two-step method showed no detectable O-methylation ((1)H NMR) and a slight increase in molecular weight with increasing DQ (GPC), implying that no chain scission occurred during synthesis. The solubility in aqueous solutions at pH 7 of O-methyl free TMC with DQ<24% was less as compared to O-methylated TMC with the same DQ. On the other hand, O-methyl free TMC with DQ>33% had a good aqueous solubility. On Caco-2 cells, O-methyl free TMCs demonstrated a larger decrease in transepithelial electrical resistance (TEER) than O-methylated TMCs. Also, with increasing DQ, an increase in cytotoxicity (MTT) and membrane permeability (LDH) was observed.