The wide-scale use of molecular phylogenetic studies in mycology has significantly changed our understanding of the relationships between fungal taxa at all levels, including species, genera, families, and beyond. After extensive evaluation in a large international barcoding consortium, the internal transcribed spacer (ITS) region was selected as the universal fungal barcode, despite the general knowledge that this marker does not distinguish between all fungal species. Multiple genes were used to improve phylogenetic resolution among ascomycetous and basidiomycetous yeasts, dermatophytes, dimorphic fungi, Aspergillus, Penicillium, and Fusarium. The most recent development is the use of whole genome-based data in phylogenomic studies. With respect to clinically important fungi, these studies have yielded several important new insights that are highlighted in this chapter.