The mechanisms responsible for the origin and maintenance of large non-recombinant regions on sex chromosomes have been studied primarily in plants and animals, but the recent discovery of similar features on fungal chromosomes carrying sex chromosomes type of mating in different species could shed new light on this phenomenon (Fraser et al. 2004). Sex chromosomes in plants and animals evolved from an autosomal pair by expansion of the nonrecombinant region around complementary genes that determine sex-specific functions (Bergero and Charlesworth 2009). Such multistep expansion of nonrecombinant regions in sex chromosomes, forming “evolutionary layers” (Lahn and Page 1999), is usually explained by the recruitment of genes that result in sexually antagonistic traits (i.e., beneficial in males and deleterious in females, or vice versa). , through selection for linkage to sex-determining genes (Rice 1987, Charlesworth 2005). However, the selective forces driving the evolution of nonrecombinant regions are likely to be different in fungi because cells of different mating types show little phenotypic differences. In heterothallic fungi, syngamy can only occur between haploid cells carrying different alleles in mating-type genes, whereas in homothallic fungi no such differences are required, allowing for universal compatibility (Billiard et al. 2011). The two major fungal phyla have different genes and organization for mating type: a single locus controls mating type in ascomycetes versus two loci in basidiomycetes (i.e. haploid cells should carry different alleles at both loci for successful mating). One of the two loci controlling mating patterns in basidiomycetes encodes pheromones and pheromone receptors involved in syngamy while the other locus...... half of the article...... Smith et al. 2004) and Cryptococcus neoformans (Wang et al. 2002). In R. toluroides, the gene encoding ste20 is surrounded by genes encoding pheromones (Coelho et al. 2008). Other genes, such as abc1, with a more elusive role in fungal mating and development, have also been found in the vicinity of genes encoding the pheromone and its receptor in R. toluroides (Coelho et al. 2008). In this study, our objectives were therefore to: 1) identify additional genes belonging to the mating-type locus in Microbotryum, in particular genes encoding pheromones and homeodomain proteins, which control all types of mating in most basidiomycetes, 2) evaluate whether the genealogies of genes in the mating-type region as well as previously proposed layer loci are consistent with the existence of evolutionary layers along mating-type chromosomes.