The gene Prestin encodes a motor protein that is thought to confer the high-frequency sensitivity and selectivity that characterizes the mammalian auditory system. Recent research shows that the Prestin gene has undergone a burst of positive selection on the ancestral branch of the Old World horseshoe and leaf-nosed bats (Rhinolophidae and Hipposideridae, respectively), and also on the branch leading to echolocating cetaceans. Moreover, these two groups share a large number of convergent amino acid sequence replacements. Horseshoe and leaf-nosed bats exhibit narrowband echolocation, in which the emitted calls are based on the second harmonic of a predominantly constant frequency (CF) component, the frequency of which is also over-represented in the cochlea. This highly specialized form of echolocation has also evolved independently in the neotropical Parnell's mustached bat (Pteronotus parnellii). To test whether the convergent evolution of CF echolocation between lineages has arisen from common changes in the Prestin gene, we sequenced the Prestin coding region (~2,212 bp, >99% coverage) in P. parnellii and several related species that use broadband echolocation calls. Our reconstructed Prestin gene tree and amino acid tree showed that P. parnellii did not group together with Old World horseshoe and leaf-nosed bats, but rather clustered within its true sister species. Comparisons of sequences confirmed that P. parnellii shared most amino acid changes with its congeners, and we found no evidence of positive selection in the branch leading to the genus of Pteronotus. Our result suggests that the adaptive changes seen in Prestin in horseshoe and leaf-nosed bats are not necessary for CF echolocation in P. parnellii.