In the last few decades, prestressing techniques have been used to build very important structures and infrastructures. Since the serviceability and the safety of prestressed reinforced concrete (PRC) elements rely on the effective state of prestressing, development of tools and procedures capable of estimating the effective prestress loss would be very useful. Amongst other techniques, dynamic identification has proved to be an economical, quick and reliable method to evaluate structural integrity. However, the influence of prestressing in the dynamic behavior of PRC elements is not completely clear. In fact, while many references in the literature state that the prestressing force does not affect the frequencies of vibration, almost every experimental test carried out on PRC beams shows an increase in the eigenfrequencies for increasing value of the prestressing force. This paper aims to contribute to the debate, investigating the dynamic behaviour of PRC beams taking into account properties, such as nonlinearity, softening, confinement and micro-cracking of concrete subjected to compression and tension stress states, and the variation of the flexural stiffness of the PRC beam along its length according to bending stress distribution. Non-linear discrete modelling, in combination with system identification and optimization was used to define the dynamic properties of PRC beams, taking into account the effect of prestressing level. The proposed model was applied to four PRC beams with known mechanical and dynamic properties in literature. The influence of the prestressing force on the trend of frequencies of vibration was closely captured for each beam, with errors less than 3% of the estimated frequencies. The results of this investigation thus indicate that dynamic identification techniques can potentially be used to identify the prestressing force level, and consequently the prestress loss, provided the complete concrete mechanics is taken into account.