26Ferns are the second most dominant group of land plants after angiosperms. Extant species occupy an 27 extensive range of habitats and contribute significantly to ecosystem functioning. Despite the 28 importance of ferns, most taxa are poorly covered by genomic resources. The genus Botrychium 29 belongs to one of the earliest divergent lineages of vascular plants and has a global distribution with 30 35 species, half of which are polyploids. Here, we establish a reference transcriptome for Botrychium 31 lunaria, a diploid species with an extremely large genome of 27.5 Gbp. We assembled 25,721 high-32quality transcripts with an average length of 1,332 bp based on deep RNA-sequencing of a single 33 individual. We sequenced an additional 11 transcriptomes of individuals from two populations in 34 Switzerland, including the population of the reference individual. Based on read mapping to reference 35 transcript sequences, we identified 374,510 single nucleotide polymorphisms (SNPs) segregating 36 among individuals for an average density of 14 SNPs per kb. The transcriptome-wide markers 37 provided unprecedented resolution of the population genetic structure revealing substantial variation in 38 heterozygosity among individuals. We also constructed a phylogenomic tree of 73 taxa representating 39 all fern orders to ascertain the placement of the genus Botrychium. The high-quality transcriptomic 40 resources enable powerful population and phylogenomic studies in an important group of ferns. 41 42 43