The mechanism of ATP hydrolysis in the myosin motor domain is analyzed using a combination of DFTB3/CHARMM simulations and enhanced sampling techniques. The motor domain is modeled in the pre-powerstroke state, in the post-rigor state, and as a hybrid based on the post-rigor state with a closed nucleotide-binding pocket. The ATP hydrolysis activity is found to depend on the positioning of nearby water molecules, and a network of polar residues facilitates proton transfer and charge redistribution during hydrolysis. Comparison of the observed hydrolysis pathways and the corresponding free energy profiles leads to detailed models for the mechanism of ATP hydrolysis in the pre-powerstroke state and proposes factors that regulate the hydrolysis activity in different conformational states. In the pre-powerstroke state, the scissile Pγ-O3β bond breaks early in the reaction. Proton transfer from the lytic water to the γ-phosphate through active site residues is an important part of the kinetic bottleneck; several hydrolysis pathways that feature distinct proton transfer routes are found to have similar free energy barriers, suggesting a significant degree of plasticity in the hydrolysis mechanism. Comparison of hydrolysis in the pre-powerstroke state and the closed post-rigor model suggests that optimization of residues beyond the active site for electrostatic stabilization and preorganization is likely important to enzyme design.