The mating pattern between 38 strains collected at various places in Europe and Southern India was determined. There are at least three compatibility groups: A (23 strains) and B (9 strains) comprising the European isolates, and C (6 strains), the Indian isolates. Within each compatibility group sexual reproduction is, as expected, controlled by a bipolar mechanism of homogenic incompatibility. No fertile offspring are obtained in any intergroup crossing showing that there is genetic separation by heterogenic incompatibility. However, the European group B seems to be closer related to the Indian (C) group in that sterile fruit bodies are produced between + and − mating types. An indication for a further subdivision is the occurrence of a barrage-like phenomenon between representatives of all three groups. The data thus indicate how the start of speciation may be occurring in Ascobolus immersus by means of both spatial and genetic isolation.