Bats, as the world’s second-largest mammalian order, have garnered significant attention for their ability to harbor numerous viruses without exhibiting disease symptoms. Nucleotide-binding domain and leucine-rich repeat-containing receptors (NLRs) are crucial components of the immune system. This study conducted an evolutionary analysis of the NLR gene family across 26 bat species to investigate the molecular mechanisms underlying their role in viral resistance under high viral load pressure. We identified gene duplication events in multiple genes. The NLR gene family exhibited high conservation throughout evolution, which may contribute to the occurrence of gene duplication. This conserved genomic structure also ensures functional stability, safeguarding bats’ antiviral resistance. Most NLR genes primarily function within the type I interferon (IFN) signaling pathway and the NF-κB signaling pathway. The NLR gene family enhances the innate immune capacity of bats through the adaptive evolution of some genes, combining enhanced gene functionality with the maintenance of gene conservation at a low evolutionary rate. Moreover, bats employ diverse innate immune strategies, where multiple immune pathways collectively establish the innate immune barrier. The molecular evolution of this gene family provides new insights into the molecular mechanisms and functional pathways involved in the innate immune response of bats.