Prestressed concrete structures are considered to be reliable and durable. However, their long-term performance when subjected to frost attack is still unclear. In this work, experiments were carried out to evaluate the prestress losses in post-tensioned prestressed concrete specimens subjected to freeze–thaw cycles (FTCs). Two cases were considered: in one case, a series of specimens were prepared and tested in a freeze–thaw chamber; in the second case, the same series of specimens were tested in an indoor environment (outside the chamber). The difference between the prestress losses of the specimens inside the freeze–thaw chamber and those outside the chamber equalled the prestress losses due to FTCs. When using mathematical models to predict the prestress losses due to the FTCs, it was found that they were relatively small when the concrete was slightly damaged. However, they increased rapidly when the FTCs were repeated. The eccentricity of the prestress wires led to larger prestress losses when subjected to FTCs. Moreover, the same cross section and eccentricity resulted in similar prestress losses due to the FTCs, and the relatively high-strength concrete could withstand more FTCs.