Abstract: Horseshoe bats (genus Rhinolophus, family Rhinolophidae) represent an important group within the bat families. These bats have several notable characteristics that distinguish them from other bats, such as the specialized high constant frequency echolocation calls, rapid karyotype evolution, as well as the increasing reports about the their unique immune system. Evolutionary studies supported by high-quality reference genome and whole-genome data provide deeper insights into species origin, speciation, adaptive evolution, and phenotypic variation. However, so far, the genomic data limitation of only one published genome of the horseshoe bat, R. ferrumequinum, creates obstacles for a deeper understanding of the evolutionary patterns in Rhinolophus. In this research, we constructed a high-quality chromosome-level reference genome for the intermediate horseshoe bat (R. affinis). Our comparative genomic analyses revealed potential genetic characteristics associated with virus tolerance in Rhinolophidae. This includes the expansion of several gene families related to immune response, as well as the identification of genes functionally associated with the SARS-CoV-2 signaling pathway, DNA repair, and apoptosis that exhibit signs of rapid evolution. In addition, we observed an expansion of the MHC-2 region and a higher copy number of the HLA-DQB2 gene in horseshoe bats compared to other bats. By generating whole-genomic resequencing data for population genomic analyses, we identified multiple candidate loci (e.g. GLI3) associated with echolocation call frequency variation across R. affinis subspecies. Our work has expanded the understanding of the genetic characteristics of the horseshoe bat group, while also provides a data foundation for future research.