Limber pine (Pinus flexilis) is a keystone species of high-elevation forest ecosystems of western North America, but some parts of the geographic range have high infection and mortality from the non-native white pine blister rust caused by Cronartium ribicola. Genetic maps can provide essential knowledge for understanding genetic disease resistance as well as local adaptation to changing climates. Exome-seq was performed to construct high-density genetic maps in two seed families. Composite maps positioned 9612 unigenes across 12 linkage groups (LGs). Syntenic analysis of genome structure revealed that the majority of orthologs were positional orthologous genes (POGs) with localization on homologous LGs among conifer species. Gene ontology (GO) enrichment analysis showed relatively fewer constraints for POGs with putative roles in adaptation to environments and relatively more conservation for POGs with roles in basic cell function and maintenance. The mapped genes included 639 nucleotide-binding site leucine-rich repeat genes (NBS-LRRs), 290 receptor-like protein kinase genes (RLKs), and 1014 genes with potential roles in the defense response and induced systemic resistance to attack by pathogens. Orthologous loci for resistance to rust pathogens were identified and were co-positioned with multiple members of the R gene family, revealing the evolutionary pressure acting upon them. This high-density genetic map provides a genomic resource and practical tool for breeding and genetic conservation programs, with applications in genomewide association studies (GWASs), the characterization of functional genes underlying complex traits, and the sequencing and assembly of the full-length genomes of limber pine and related Pinus species.