Berkeleyomyces rouxiae, a soil-borne fungal pathogen, causes black root rot on various plant species including carrot, okra, tobacco, lettuce, and other important crops. Here we analyzed the entire genome sequence of a Japanese isolate of B. rouxiae and discovered 25 microsatellite sequences. These sequences were amplified from the genomic DNA of several Japanese isolates by PCR and sequenced, revealing that the microsatellite sequences were polymorphic among isolates. These markers are useful for phylogenetic analysis, epidemiological population analysis, identification of specific isolates, and other genetic investigation of B. rouxiae.