Two bis(imidazole) derivatives, 1,4-di(1H-imidazol-1-yl) butane dihydrate (DImB·2H2O) and 1,4-di(1H-2-methylimidazol-1-yl) butane tetrahydrate (DMImB·4H2O were synthesized. The crystallization was performed from an aqueous ethanol by slow evaporation technique. X-ray crystallography was used to confirm the molecular structures of both crystals. The results from crystal structure analysis displayed that DImB·2H2O was crystallized in the orthorhombic crystal system and Pbca space group with a = 14.7805(4) Å, b = 5.22600(10) Å, and c = 15.6110(4) Å. The molecule lies in the center of inversion, making the asymmetric unit consist of half of the parent molecule with one unit of the water molecule, whereas DMImB·4H2O displayed the triclinic crystal system and R-3 space group with a = 21.526(3) Å, b = 21.526(3) Å, and c = 9.3333(12) Å. The molecule lies on a center of inversion, making the asymmetric molecules consist of half of the parent molecule with two units of the water molecules. The effect of replacing hydrogen atoms at C2 of two imidazole rings with the methyl groups on spectroscopic properties was investigated using FTIR, MS, 1H, and 13C NMR. The infrared spectrum demonstrated the presence of water molecules in DMImB·4H2O, which showed a more intense O‒H group peak than DImB·2H2O. The effect of methyl groups was studied on the chemical shifts, multiplicity, and coupling constants of hydrogen atoms, and carbon chemical shifts of imidazole rings. Mass spectra of both compounds showed [M+Na]+ and [2M+Na]+ in positive ion mode (ESI+). In addition, the thermal phase transitions and thermal stability of the bis(imidazole) derivatives were studied using the DSC and TGA/DTG. The crystalline structures of DImB and DMImB recorded and the methyl effect was investigated on the size of molecules, arrangement of molecules in the crystal lattice, lattice planes, solvent void, length, angle, and torsion.