Here, we report the use of the mating type heterokaryon incompatibility system as a counterselection to increase the probability of identifying gene replacements in Neurospora crassa. We compared the frequencies of gene replacements observed among transformants obtained by using plasmids with or without the mat a-1(+) gene (hereby called "Toxic Gene") placed adjacent to disruption cassettes. On an average, we were 20x more likely to identify a correct gene replacement by incorporating the toxic gene in our constructs. Using this strategy, we constructed strains containing a deletion of the inl (1L-myo-inositol-1-phosphate synthase) gene. Finally, we demonstrated that we were able to remove the transformation marker (the hygromycin B phosphotransferase- thymidine kinase gene fusion [hph(+)::tk(+)]) from the genome by using a strategy similar to the "URA-blaster" strategy used in yeast, which we call "tk-blaster."