In the last decades, research efforts have been spent to investigate the effect of prestressing on the dynamic behaviour of prestressed concrete (PSC) beams. Whereas no agreement has been reached among the achievements obtained by different Researchers and among the theoretical and the experimental results for simply supported beams, very few researches have addressed this problem in continuous PSC beams. This topic is, indeed, worthy of consideration bearing in mind that many relevant bridges and viaducts in the road and railway networks have been designed and constructed with this structural scheme. In this paper the attention is, thus, focused on the dynamic features of continuous PSC bridges taking into account the effect of prestressing. This latter, in fact, contributes to the modification of the distribution of the bending stress along the beam, also by means of the secondary moments, and influences the flexural stiffness of the beam itself. The dynamic properties of a continuous, two spans bridge connected by a nonlinear spring have been extracted by solving an eigenvalue problem in different linearized configurations corresponding to different values of the prestress force. The stiffness of the nonlinear spring has been calculated considering the mechanical behaviour of the PSC beam in the uncracked and in the cracked stage. The application of the proposed methodology to several case studies indicates that the shift from the uncracked to the cracked stage due to an excessive prestress loss is clearly detectable looking at the variation of the dynamic properties of the beam. In service conditions, this shift happens for low values of the prestress losses (up to 20%) for structure with a high value of the ratio between the permanent load and the total load, as happens for instance in long span, continuous box bridges. In such conditions, the detection of the dynamic properties can provide meaningful information regarding the structural state of the PSC beam.