Flammulina velutipes, amongst others known as Winter Mushroom or Enokitake is an important economic crop in Asia. The tetrapolarity (having four mating types) of this mushroom obligates mating and results in self-sterile progeny that carries unique genetic traits, making understanding of the genetic base desirable for breeding. Moreover, mating type genes are significant for evolutionary studies as their high polymorphism benefits phylogenetic comparisons. This polymorphism further makes mating type genes interesting candidates for genetic markers that allow identification of specific strains. Mating type loci in Agaricomycotina are classically termed A and B and control two different developmental pathways [for a review see 1]. They consist of tightly linked subloci that encode multiallelic genes. MatA loci contain two groups of divergently transcribed homeodomain proteins (HD1 and HD2) and heterodimerization of HD1 with a non-self HD2 protein forms a functional transcription factor that activates the A pathway. MatB loci hold pheromones and pheromone receptors. Pheromone genes encode small precursor proteins that are characterized by a C-terminal CAAX motif. Pheromone receptors typically contain 7 membrane spanning regions and are coupled to G-proteins. Binding of a pheromone to a receptor, triggers splicing of the (trimeric) G-protein, which activates the B pathway. New genetic data from recent genome sequences is challenging the strict concepts of old mating type models in fungi. MatB loci turn out to be rather diverse and contain considerable varying numbers of pheromone receptors and associated pheromones. To this, pheromone receptors which are not linked to matB loci have now been reported for C. cincerea, S. commune and L. bicolor [2, 3]. Also the organization of the matA locus is less strictly conserved than anticipated. Though far more tightly maintained than the matB locus, substantial differences in HD gene numbers and overall organization are reported [2, 3]. These differences stress the importance of determination of the individual mating type systems from industrially important mushrooms to assist breeding. Our analysis of F. velutipes strain 4019-20 uncovered 7 pheromone receptors together with 3 pheromones. The matB-3 locus of this strain however, is defined by only a single pheromone receptor and pheromone gene and our data strongly indicates that a 2nd pheromone receptor recently lost its function. The other receptor genes are non mating type specific. Finally, we detected three homeodomain genes distributed over two distant subloci. These subloci have been separated by two large inversions. Strikingly the distant matA subloci in S. commune seem to be separated by inversions as well. Synthenic mapping of a large regions from Coprinus cinerea, Laccaria bicolor, S. commune and F. velutipes shows that the matA loci originate from a single locus in a common ancestor of S. commune and F. velutipes that is represented by L. bicolor and C. cinerea. [1] U Kües. Micr Mol Biol Rev 64, 316 (2000) [2] H Niculita-Hirzel, J Labbé, A Kohler, F le Tacon, F Martin, IR Sanders and U Kües. New Phytol 180, 329 (2008). [3] RA Ohm, JF de Jong, LG Lugones, A aerts, E Kothe, JE Stajich, RP de Vries, E Record, A Levasseur, SE Baker, KA Bartholomew, PM Couthino, S Erdmann, TJ Fowler, AC Gathman, V Lombard, B Henrissat, N Knabe, U Kües, WW Lilly, E Lindquist, S Lucas, JK Magnuson, F Piumi, M Rausdaskoski, A Salamov, J Schmutz, FWMr Schwarze, PA van Kuyk, JS Horton, IV Grigoriev and HAB Wösten. Nat. Biotechnol ISSN: 1087-0156 (2010).