Natural sounds consist of a component at the fundamental frequency (f0) and its overtones. Pitch is perceived at f0, even when spectral energy at f0 is missing. This missing f0, or ‘virtual pitch’, is thought to be detected in the auditory cortex and related cortical areas, but the precise neural mechanisms are unknown. One possibility is that virtual pitch can be retrieved from the periodicity of sound waveforms. However, this mechanism requires the temporal accuracy in periodicity detection, and so far the detection of virtual pitch has only been demonstrated at frequencies lower than 1kHz. We investigated the ability of mice to detect virtual pitch up to 5kHz using a two-step sound discrimination test. In the first step of this test, mice were trained to discriminate between tone bursts at 2.5 and 5kHz. In the second step, we tested the ability of mice to discriminate between virtual pitches at 2.5kHz and at 5kHz. It was demonstrated that the performance of mice to discriminate between virtual pitches at 2.5 and 5kHz was significantly affected by previous discrimination learning between tone bursts, indicating that mice can detect virtual pitch up to 5kHz.