Additive manufacturing (AM) is a modern way of manufacturing structures, which tends to have fewer design limitations than those manufactured by conventional processes such as casting or forging. A combination of high-strength materials and small and complex structures opens up a wide range of potential applications, especially in the fields of medicine and aerospace. Titanium and its alloys show a very beneficial combination of density and mechanical properties. One of these alloys is the metastable β titanium alloy Ti– 5Al–5Mo–5 V–3Cr (Ti-5553), which is currently used mainly for large forged structures like landing gears of airplanes. In this study, for the first time the fatigue behavior of electron beam powder bed fused (PBF-EB) Ti-5553 was investigated with a focus on the defects created by the layer wise manufacturing. To understand the defect structure and its respective influence on the fatigue behavior, all specimens were scanned prior to fatigue testing using a state-of-the-art µ-focus CT. The specimens were subjected to two heat treatment procedures commonly used in technical applications, which were aiming for high strength (solution treated and aged—STA) as well as high ductility (beta annealed, slow cooled and aged—BASCA). Results indicate that the fatigue strength of PBF-EB manufactured Ti-5553 is significantly reduced compared to conventionally manufactured Ti-5553. The main reason for this are defects, which have varying critical effects depending on the heat treatment of the specimen and the defect size, shape, location and type.