White-nose syndrome (WNS) has decimated numerous bat populations in North America. Given the rapid spread of WNS, identifying resistance is critical for mitigating further loss. Yet, detecting genomic signatures of WNS resistance in bat populations has proven challenging. Rapid innovations in sequencing and genome assembly technologies now provide unprecedented opportunity to characterize genomic variation in virtually any species and, in turn, resolve the underpinnings of wildlife disease resistance. We have assembled a near-chromosome-level for the little brown bat (Myotis lucifugus) reference genome using Omni-C and PacBio sequencing, resulting in a mean depth of coverage of 37.7X, a scaffold N50 of 99.0 Mb, and a BUSCO completeness score of 98.5%. We optimized gene annotation by combining ab initio gene predictions, orthology inferences, and transcriptomic evidence. Additionally, we conducted whole-genome resequencing on M. lucifugus samples predating, and 10 years following, WNS exposure. Individuals were sequenced from sites in New York and Pennsylvania (N ≈ 15 per sampling) at a mean depth of 25.7X resulting in >40 million SNPs. To date, this study has produced the highest-quality genomic resources available for M. lucifugus for determining the genomic architecture of WNS resistance. These findings will in turn provide insight into naïve population susceptibility during future spread of WNS.