The microstructural evolution of a thermoplastic polyurethane (TPU) with low hard segment content has been monitored utilizing in situ real-time synchrotron small angle X-ray scattering (SAXS) and time-domain nuclear magnetic resonance (NMR) measurements. The TPU is composed of 23 wt% of [4,4-methylenediphenyl diisocyanate (MDI)]-[1,4-butanediol (BD)] chain segments, which form hard domains, as [polytetrahydrofuran (PTHF)] forming soft domains. The number and distribution of monomer units in hard blocks is determined by the successive self-nucleation and annealing thermal fractionation technique. In situ SAXS method reveals heating-induced increase in the spacing of hard and soft domains, while time-domain 1H-NMR characterizes the changes in the phase composition and chain dynamics in these domains. A glassy fraction of short MDI-BD chain segments in hard domains passes through Tg above ambient temperature. At higher temperatures, MDI-BD nanocrystals start to melt. Sequence length distribution of MDI-BD chain segments causes a distribution in crystal sizes and wide melting temperature range. The melting is accompanied by the mixing of MDI-BD with PTHF segments in soft domains, and by increase in segmental mobility in these domains. Above 180 °C, the TPU melt is homogeneous on the scale above nanometers according to SAXS data. [ABSTRACT FROM AUTHOR]