The ability to mate fungi in the laboratory is a valuable tool for genetic analysis and for classical strain improvement. In ascomycetous fungi, mating typically occurs between morphologically identical partners that are distinguished by their mating type. In most cases, the single mating-type locus conferring mating behavior consists of dissimilar DNA sequences (idiomorphs) in the mating partners. All ascomycete mating-type idiomorphs encode proteins with confirmed or putative DNA-binding motifs. These proteins control, as master regulatory transcription factors, pathways of cell speciation and sexual morphogenesis. Mating-type organization of four of the six classes of ascomycetes has been studied at the molecular level over the past 20 years. This review gives a short overview of the structural organization of the mating-type loci of yeasts and filamentous ascomycetes. In addition, this review describes how the availability of mating-type sequences allows the investigation of key issues concerning genetic and phylogenetic analyses of fungal species.