<p>The fatigue resistance of casting polyurethane (CPU) is crucial in various sectors, such as construction, healthcare, and the automotive industry. Despite its importance, no studies have reported on the fatigue threshold of CPU. This study employed an advanced Intrinsic Strength Analyzer (ISA) to evaluate the fatigue threshold of CPUs, systematically exploring the effects of three types of isocyanates (PPDI, NDI, TDI) that contribute to hard segment structures based on the cutting method. Employing multiple advanced characterization techniques (XRD, TEM, DSC, AFM), the results indicate that PPDI-based polyurethane exhibits the highest fatigue threshold (182.89 J/m²) due to a highest phase separation and a densely packed spherulitic structure, although the hydrogen bonding degree is the lowest (48.3%). Conversely, NDI-based polyurethane, despite having the high hydrogen bonding degree (53.6%), exhibits moderate fatigue performance (122.52 J/m²), likely due to a more scattered microstructure. TDI-based polyurethane, with the highest hydrogen bonding degree (59.1%) but absence of spherulitic structure, shows the lowest fatigue threshold (46.43 J/m²). Compared to common rubbers (NR, NBR, EPDM, BR), the superior fatigue performance of CPU is attributed to its well-organized microstructure, polyurethane possesses a higher fatigue threshold due to its high phase separation degree and orderly and dense spherulitic structure which enhances energy dissipation and reduces crack propagation.</p>