Aim To develop a pyrosequencing assay to monitor the frequency of alleles of an avirulence gene, AvrLm4, in populations of sexual spores of Leptosphaeria maculans, a fungal pathogen of canola (Brassica napus). Methods and Results The predominant mutation in AvrLm4 responsible for virulence to the corresponding resistance gene, Rlm4, is a single nucleotide polymorphism (SNP) at base 358. Pyrosequencing primers were designed to amplify a 90-bp region that included this SNP. The assay was developed and validated by analysing the frequency of AvrLm4 in isolate mixtures of different proportions. Furthermore, the frequency of avrLm4 (virulence allele) determined by pyrosequencing of populations of sexual spores was consistent with the frequency of avrLm4 determined by Sanger sequencing of the entire AvrLm4 gene from single isolates cultured from the same stubble. Conclusion This high-throughput assay can play an important role in predicting the risk of resistance breakdown in crops. Significance and Impact of the Study Similar assays can be applied to monitor frequencies of fungicide resistance in pathogens of crops and to assay diversity in microbial soil communities such as in soil samples from bat caves where white-nose syndrome has been detected.