Geckos use vocalizations for intraspecific communication, but little is known about the organization of their central auditory system. We therefore used immunohistochemical techniques to delineate the auditory nuclei of the hindbrain, midbrain and thalamus. We used antibodies against the calcium binding proteins calretinin (CR), parvalbumin (PV), calbindin-D28k (CB) as well as antibodies against Glutamic Acid Decarboxylase (GAD) and synaptic vesicle protein 2 (SV2). Western blots showed that all five antibodies were specific within gecko brain. The fluorescence double labeling of calcium binding proteins yielded partly overlapping but mostly complementary distributions in the gecko auditory pathway. The differential expression of all calcium binding proteins and GAD, as well as SV2, characterized the structures of the gecko auditory system. The results indicate that the auditory structures and organization in Gekko gecko bear many similarities to the other land vertebrates.