Differences in gene expression within tissues can lead to differences in tissue function. Understanding the transcriptome of a species helps elucidate the molecular mechanisms underlying phenotypic divergence. According to the presence or absence of a reference genome of for a studied species, transcriptome analyses can be divided into reference‑based and reference‑free methods, respectively. Presently, comparisons of complete transcriptome analysis results between those two methods are still rare. In this study, we compared the cochlear transcriptome analysis results of greater horseshoe bats (Rhinolophus ferrumequinum) from three lineages in China with different acoustic phenotypes using reference‑based and reference‑free methods to explore their differences in subsequent analysis. The results gained by reference-based results had lower false-positive rates and were more accurate because differentially expressed genes among the three populations obtained by this method had greater reliability and a higher annotation rate. Some phenotype-related enrichment terms, including those related to inorganic molecules and proton transmembrane channels, were also obtained only by the reference-based method. However, the reference‑based method might have the limitation of incomplete information acquisition. Thus, we believe that a combination of reference‑free and reference‑based methods is ideal for transcriptome analyses. The results of our study provided a reference for the selection of transcriptome analysis methods in the future.