The membrane motor protein, prestin, encoded by gene Prestin, provides the electromotility to cochlear outer hair cells (OHCs) and is therefore considered responsible for cochlea's high sensitivity to sound waves. Echolocating bats use ultrasound for orientation and hunting. In this study, we obtained the complete Prestin coding sequences of 60 mammal taxa. Alignment results detected the same 3-bp deletion mutation (c.1833_1835del in exon 18) in two bat lineages, Pteropodidae and Emballonuroidea, and caused an amino acid deletion (p.Asp611del). These two bat lineages orient with vision and echolocation, separately, suggesting that the parallel deletion occurred independently after their split. Homology modeling of the protein structures indicated contrasting structural variations after the deletion of this amino acid. Estimation of the distributions of the surface electrostatic potential coincided with the structural variation. Our findings suggest the complexity of the echolocation in bats but functionality analyses are needed to illuminate it.