Despite the critical roles fungi play in the functioning of ecosystems, especially as symbionts of plants and recyclers of organic matter, their biodiversity is poorly known in high-latitude regions. In this paper, we discuss the molecular diversity of one of the most diverse and abundant groups of ectomycorrhizal fungi: the genus Lactarius Pers. We analysed internal transcribed spacer rDNA sequences from both curated sporocarp collections and soil polymerase chain reaction clone libraries sampled in the arctic tundra and boreal forests of Alaska. Our genetic diversity assessment, based on various phylogenetic methods and operational taxonomic unit (OTU) delimitations, suggests that the genus Lactarius is diverse in Alaska, with at least 43 putative phylogroups, and 24 and 38 distinct OTUs based on 95% and 97% internal transcribed spacer sequence similarity, respectively. Some OTUs were identified to known species, while others were novel, previously unsequenced groups. Non-asymptotic species accumulation curves, the disparity between observed and estimated richness, and the high number of singleton OTUs indicated that many Lactarius species remain to be found and identified in Alaska. Many Lactarius taxa show strong habitat preference to one of the three major vegetation types in the sampled regions (arctic tundra, black spruce forests, and mixed birch-aspen-white spruce forests), as supported by statistical tests of UniFrac distances and principal coordinates analyses (PCoA). Together, our data robustly demonstrate great diversity and nonrandom ecological partitioning in an important boreal ectomycorrhizal genus within a relatively small geographical region. The observed diversity of Lactarius was much higher in either type of boreal forest than in the arctic tundra, supporting the widely recognized pattern of decreasing species richness with increasing latitude.