The relationship between a microphase-separated structure and dynamic viscoelastic properties of polyurethane elastomers (PUEs) prepared at various temperatures and cross-linking agent contents were investigated. The PUEs were synthesized with poly(oxytetramethylene) glycol (PTMG), 4,4'-diphenylmethane diisocyanate (MDI) and 1,4-butanediol (BD) and a mixture of BD and 1,1,1-trimethylol propane (TMP) by a prepolymer method. The PUEs are in the strong-segregation limit. The microphase-separated structure was investigated using Fourier-transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), small-angle x-ray scattering (SAXS) and atomic force microscope (AFM). The extent of microphase separation of the PUEs became stronger and weaker with increases in curing temperature and trifunctional TMP content, respectively. The consistent results obtained in this study would be quite helpful to design the desirable PUEs.