Genetic recombination is an important tool in strain breeding in many organisms. We studied the possibilities of mitotic recombination in strain breeding of the asexual fungus Aspergillus niger. By identifying genes that complemented mapped auxotrophic mutations, the physical map was compared to the genetic map of chromosome III using the genome sequence. In a program to construct a chromosome III-specific marker strain by selecting mitotic crossing-over in diploids, a mitotic recombination hotspot was identified. Analysis of the mitotic recombination hotspot revealed some physical features, elevated basal transcription and a possible correlation with purine stretches.