Cyclic fatigue experiments in the high and very high cycle fatigue regimes have been performed on a René 88DT polycrystalline nickel-based superalloy. The microstructural configurations that favor early strain localization and fatigue crack initiation at high temperature from 400 °C to 650 °C have been investigated. Competing failure modes are observed in the high to the very high cycle fatigue regime. Fatigue cracks initiate from non-metallic inclusions and from intrinsic internal microstructural features. Interestingly, as stresses are reduced into the very high cycle regime, there is a transition to initiation only at crystallographic facets. At higher stress in the high cycle fatigue regime, a significant fraction of specimens initiate cracks at non-metallic inclusions. This transition is analyzed with regard to microstructural features that favor strain localization and accumulate damage early during cycling.