This paper presents a probabilistic framework for updating the structural reliability of offshore wind turbine substructures based on digital twin information. In particular, the information obtained from digital twins is used to quantify and update the uncertainties associated with the structural dynamics and load modeling parameters in fatigue damage accumulation. The updated uncertainties are included in a probabilistic model for fatigue damage accumulation used to update the structural reliability. The updated reliability can be used as input to optimize decision models for operation and maintenance of existing structures and design of new structures. The framework is exemplified based on two numerical case studies with a representative offshore wind turbine and information acquired from previously established digital twins. In this context, the effect of updating soil stiffness and wave loading, which constitute two highly uncertain and sensitive parameters, is investigated. It is found that updating the soil stiffness significantly affects the reliability of the joints close to the mudline, while updating the wave loading significantly affects the reliability of the joints localized in the splash zone. The increased uncertainty related to virtual sensing, which is employed to update wave loading, reduces structural reliability.