This article describes a simple, reliable, efficient, and general method for the synthesis of 7-methylguanosine nucleotides such as 7-methylguanosine 5'-O-monophosphate (m7 GMP), 7-methylguanosine 5'-O-diphosphate (m7 GDP), 7-methyl-2'-deoxyguanosine 5'-O-triphosphate (m7 2'dGTP), and 7-methylguanosine 5'-O-triphosphate (m7 GTP) starting from the corresponding guanosine nucleotide is described. The present protocol involves methylation reaction of guanosine nucleotide using dimethyl sulfate as a methylating agent and water as a solvent at room temperature to provide the corresponding 7-methylguanosine nucleotide in good yields with high purity (>99.5%). It is noteworthy that the present methylation reaction proceeds smoothly under aqueous conditions that is highly regioselective to afford exclusive 7-methylguanosine nucleotide. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Synthesis of 7-methylguanosine nucleotides.