Lactifluus is a distinct genus of milkcaps, well known as ectomycorrhizal fungi. The characteristics of the genus Lactifluus include grayish-yellow, orange to orange-brown, or reddish-brown pileus, white latex from the damaged lamellae, discoloring to a brownish color, reticulate spore ornamentation, lampropalisade-type pileipellis, and the presence of lamprocystidia. Guizhou Province is rich in wild mushroom resources due to its special geographical location and natural environment. In this study, three novel Lactifluus species were identified through the screening of extensive fungal resources in Suiyang County, Guizhou Province, China, sampled from host species of mostly Castanopsis spp. and Pinus spp. Based on critical morphology coupled with nuclear sequences of genes encoding large subunit rRNA, internal transcribed spacer, and RNA polymerase II, these new species, Lactifluus taibaiensis, Lactifluus qinggangtangensis, and Lactifluus jianbaensis, were found to belong to Lactifluus section Lactifluus. A comparison with closely related species, Lactifluus taibaiensis was distinguished by its lighter-colored pileus, different colors of lamellae, and more subglobose basidiospores; Lactifluus jianbaensis was identified by the height of the spore ornamentation and its subglobose basidiospores; and Lactifluus qinggangtangensis was characterized by having smaller basidiospores, ridges, and pleurolamprocystid.