The genus Lactifluus is one of the common ectomycorrhizal fungal taxa in tropical African forest ecosystems. Recent morphological and anatomical mycological studies based on specimens we sampled from 2007 to 2013 in West African forest ecosystems, including dry, dense, riparian forests and woodlands, enable to assess the diversity and the occurrence of Lactifluus species in the Guineo-Sudanian domain. A total of 51 ITS rDNA sequences generated from our samples were aligned against tropical African and worldwide Lactifluus sequences available in GenBank. A Maximum Likelihood phylogenetic tree was inferred from 113 sequences. The phylogenetic placement of the species, combined with our morpho-anatomical data, supported the description of five new species distributed among Lactifluus species. Our data further confirm that the species richness of the genus Lactifluus is high and partly unexplored in the Guineo-Sudanian domain, and confirmed that, in both the Guineo-Sudanian and the Congo-Zambezian domain many common species occur. Patterns of occurrence of the recorded Lactifluus species from Guineo-Sudanian ecozones are also highlighted.