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The process of crack initiation has been investigated in three widely used titanium alloys with different microstructures and loading conditions. Using low-cycle fatigue tests, a unique crack nucleation mechanism involving strain localization at ( 0001 ) twist boundaries has been identified. In order to constitute a potential crack initiation site, the twist boundary must experience a high resolved shear stress and a high normal stress. Crack initiation at these boundaries is most frequently associated with twist angles spanning the 10° - 20° range. Deformation prior to crack initiation at these rare microstructural configurations has been characterized using transmission electron microscopy and high-resolution digital image correlation across large fields of view. The ( 0001 ) twist boundaries are preferential locations for early and intense strain localization. Prior to crack nucleation, deformation proceeds via shear along such boundaries where no β layer at the interface was evidenced. The presently discussed crack formation mechanism is believed to be of broad relevance as it is not significantly influenced by microstructural parameters such as the α grain size, the degree of microtexture, the β phase fraction or the surrounding microstructure as well as α and β compositions.