Abstract The structure of the mammalian auditory brainstem is evolutionarily highly plastic, and distinct nuclei arrange in a species‐dependent manner. Such anatomical variability is present in the superior olivary complex (SOC) and the nuclei of the lateral lemniscus (LL). Due to the structure–function relationship in the auditory brainstem, the identification of individual nuclei supports the understanding of sound processing. Here, we comparatively describe the nucleus arrangement and the expression of functional markers in the auditory brainstem of the two bat species Phyllostomus discolor and Carollia perspicillata . Using immunofluorescent labeling, we describe the arrangement and identity of the SOC and LL nuclei based on the expression of synaptic markers (vesicular glutamate transporter 1 and glycine transporter 2), calcium‐binding proteins, as well as the voltage‐gated ion channel subunits Kv1.1 and HCN1. The distribution of excitatory and inhibitory synaptic labeling appears similar between both species and matches with that of other mammals. The detection of calcium‐binding proteins indicates species‐dependent differences and deviations from other mammals. Kv1.1 and HCN1 show largely the same expression pattern in both species, which diverges from other mammals, indicating functional adaptations in the cellular physiology of bat neurons.