Structural dynamic properties like frequencies and mode shapes have been widely adopted for bridge condition assessment and damage identification. One of the main challenges lies in environmental factors, particularly the varying temperature, which significantly influences the bridge’s natural frequencies. In some cases, the effect of temperature changes can be comparable to, or even exceed, the effect caused by damage, rendering the damage identification methods ineffective. This study investigates the influence of the cross-sectional non-uniform temperature distribution, as a result of surrounding environmental factors, on the frequency of concrete girder bridges. A theoretical analysis is conducted to derive the bridge’s frequencies considering the cross-sectional non-uniform temperature distribution. The upper and lower bounds of the frequencies are developed by using only the environmental temperature and the largest temperature gradient defined by codes. More importantly, the damage to the bridge can be detected if the frequencies exceed the bounds for a certain period. This approach is convenient and practical in real applications without embedding thermocouples in the main girder. Numerical simulations, laboratory experimental tests, and field measurements are carried out to validate the derived frequency considering the cross-sectional non-uniform temperature distribution as well as the upper and lower bounds of the frequency. In particular, the results of the Z24 bridge show that when the bridge is damaged, the measured frequency exceeds the bound continuously, verifying the capability of the developed upper and lower bounds to evaluate bridge conditions.