Pulsed field gel electrophoresis can determine karyotypes (chromosome size and number) of fungi that cannot be studied using cytological methods, due to their chromosomes failing to condense or being too small to visualise. This technique allows genome size and genetic maps to be developed for fungi that are not amenable to classical linkage analysis. Pulsed field gel electrophoresis has revealed that many fungal plant pathogens have highly variable genomes, even within individual species and that novel-sized chromosomes can be produced in some fungi during meiosis. It can also be exploited to demonstrate the presence of dispensable (B-type) chromosomes in fungi and it is a valuable tool in taxonomic studies for distinguishing between morphologically similar fungi. Pulsed field gel electrophoresis can be used to characterise complex loci, such as those controlling host-specific toxin production in Cochliobolus species. The application of this technique to Australian isolates of Leptosphaeria maculans, which causes blackleg disease of canola (Brassica napus), is discussed.