Closely related species have been used as representative systems to investigate the genetic mechanisms involved in the early stages of species differentiation. Previous studies have indicated that variation in gene expression might be a sensitive indicator of initial species divergence, although the role of expression divergence, and especially that associated with phenotypic variation remained relatively undefined. For three organs (cochlea, brain, and liver) from two closely related bat species (Rhinolophus siamensis and R. episcopus), the interspecific and intersubspecific gene expression profiles were compared using transcriptomics in this study. Striking organ specificity of expression was observed, and expression profiles exhibited similarities between cochlea and brain tissues. Numerous differentially expressed genes (DEGs) were identified for each organ in the interspecific comparison (cochlea/brain/liver: 1,069/647/692) and intersubspecific comparison (608/528/368). Functional enrichment analysis indicated vital variation in expression related to the immune system, ion activities, neuronal function, and multisensory system regulation in both comparisons. DEGs relevant to the variation in echolocation calls (RF) were found, and some of them were involved in the pivotal patterns of expression variation. The regulation of immune, ion channel, neural activity, and sophisticated sensory functions at the expression level might be key mechanisms in the early species divergence of bats, and the expression variation related to acoustical signal could have played a crucial part. This study expands our knowledge of gene expression and patterns of variation for three key organs to echolocation at both the interspecific and intersubspecific levels. Further, the framework described here provides insight into the genetic basis of phenotypic variation during the incipient stage of species differentiation.