A perilymphatic fistula (PLF) is a rare condition in which perilymph leaks from the cochlea or vestibule into the surrounding cavities, most commonly through round and oval windows, and causes cochlear and vestibular symptoms. However, vague symptoms and the lack of a clear diagnostic test have made the existence of PLF a controversial subject for decades. Here, we report a case of definite PLF confirmed by surgery in a patient who underwent mastoidectomy 20 years prior, revealing a specific sign of missing perilymph on MRI. T2-weighted MRI revealed a nodular bright signal in the cochlea and vestibule and a large area of bright signal in the middle ear cavity with a long tail running toward the vestibule in the left ear. MRI via T2-sampling perfection with application-optimized contrasts by using a flip angle evolution sequence revealed a bright signal in the cochlear endolymph but not in the perilymph. The specific sign of a missing perilymph on MRI has a diagnostic role for PLF.