This paper presents a multi-phase model update approach for system identification of prestressed concrete (PSC) girders under various prestress forces. First, a multi-phase model update approach designed on the basis of eigenvalue sensitivity concept is newly proposed. Next, the proposed multi-phase approach is evaluated from controlled experiments on a lab-scale PSC girder for which forced vibration tests are performed for a series of prestress forces. On the PSC girder, a few natural frequencies and mode shapes are experimentally measured for the various prestress forces. The corresponding modal parameters are numerically calculated from a three-dimensional finite element (FE) model which is established for the target PSC girder. Eigenvalue sensitivities are analyzed for potential model-updating parameters of the FE model. Then, structural subsystems are identified phase-by-phase using the proposed model update procedure. Based on model update results, the relationship between prestress forces and model-updating parameters is analyzed to evaluate the influence of prestress forces on structural subsystems.