Species delimitation in Lipomyces was attempted by nuclear genome comparison in conjunction with the re-evaluation of 48 physiological characters of 65 strains. High intraspecific (> 75%) and low interspecific (< 28%) similarity values established that L. japonicus, L. lipofer and L. tetrasporus are genetically isolated, and also distinct from L. kononenkoae and L. starkeyi. Ambiguous similarity values were obtained with L. kononenkoae and L. starkeyi. Strains previously assigned to L. kononenkoae constitute two related clusters. While similarity values within each cluster range from 76-99%, representatives of the two clusters reassociate for only 47%. Since these clusters are differentiated by their ecologically relevant maximum growth temperature, L. kononenkoae is subdivided. Strains previously assigned to L. starkeyi resolve into four closely related clusters. While similarity values within each cluster range from 78-100%, representatives of the four clusters reassociate for only 59-69%. Since these four clusters are poorly differentiated, the subdivision of L. starkeyi does not appear possible without recourse to other criteria. Four unassigned strains constitute a further two clusters. Reassociation within these clusters is of the order of 91-100%, while reassociation between them occurs only at 59%. Reassociation of representatives of these clusters with those of the L. kononenkoae and L. starkeyi complexes is around 40% and 31%, respectively. These two clusters consequently appear to be intermediate between L. kononenkoae and L. starkeyi, and will, as such, have to be considered in any delimitation of these two species. A key to the taxa of Lipomyces and related genera of the Lipomycetaceae is given.