Aims: To investigate the genetic diversity among strains of Debaryomyces hansenii and further to evaluate chromosome polymorphism determined by pulse-field gel electrophoresis (PFGE) as a tool for strain typing. Methods and Results: In total 56 isolates of D. hansenii were analysed by PFGE. The isolates included type strains and other strains obtained from culture collections as well as strains collected during production of Danish surface-ripened cheeses. By use of the PFGE technique the number and size of the chromosomal bands were calculated and the total genome size estimated. The number of chromosomal bands observed was found to vary from five to 10. The most common chromosome number was found to be six and for strains with six chromosomes the total genome size was found to vary from 9.4 to 12.6 Mb. The chromosome numbers for the type strain of each variety of D. hansenii (D. hansenii var. hansenii and D. hansenii var. fabryi) appeared to be six and seven respectively. By use of the PFGE technique it was possible to differentiate between all the investigated CBS strains and the vast majority of the dairy isolates. The dairy isolates that were found to have identical profiles (three of 56 isolates) were all isolated during production of one batch of surface-ripened cheeses and are likely to be the same strain isolated several times during cheese production. Further it was shown that the PFGE analysis did not result in a division of the two D. hansenii varieties, i.e. D. hansenii var. fabryi and D. hansenii var. hansenii into separate groups. Conclusion: The present study shows that the chromosomal arrangement of D. hansenii strains is heterogenic and does have a distinct chromosome polymorphism. Further the PFGE technique was proved to have a high discriminative power for strain typing of D. hansenii. Significance and Impact of the Study: The results obtained add to the first knowledge on the genetic diversity of the species D. hansenii. Further the distinct chromosome polymorphism of D. hansenii strains as shown in this study makes the PFGE technique a useful tool for strain typing of D. hansenii, e.g. during cheese production.