Sex chromosomes and mating-type chromosomes can carry large regions with suppressed recombination. In these non-recombining regions, recessive deleterious mutations are expected to occur, as i) they are predicted to accumulate as a result of lower efficacy of selection, and ii) they may even pre-exist and drive the evolution of recombination suppression. Multiple genomic analyses have indirectly inferred the presence of deleterious mutations in sex and mating-type chromosomes, but direct experimental evidence remains scarce. Here, we performed fitness assays in fungi with megabase-large and young nonrecombining regions around the mating-type locus, using the Schizothecium tetrasporum and Podospora anserina species complexes, to test whether heterokaryons (diploid-like, heterozygous at the mating-type locus) exhibited a fitness advantage over homokaryons (haploid-like, with a single mating-type allele), in terms of spore germination dynamics or mycelium growth speed, under different conditions of light and temperature. We found a faster growth of heterokaryons compared to one of the homokaryons for P. anserina at 18°C, for S. tetrasporum and S. tritetrasporum at 22°C under light, and also at 22°C in the dark for S. tetrasporum. These findings suggest the presence of a sheltered load, i.e. recessive deleterious mutations at the heterozygous state in or near non-recombining regions, as these species are highly homozygous otherwise. Leveraging on the experimental assets of fungi, allowing cultivating separately haploid-like and diploid-like life stages, our experiments provided one of the rare direct experimental evidence of sheltered load around matingcompatibility loci, which is crucial for our understanding of sex-related chromosome evolution.