Ceratocystis fimbriata is a serious wilt and canker stain pathogen with a wide geographical distribution and host range that includes both woody and herbaceous plants. Previous studies using hybridization have shown that isolates of C. fimbriata from different hosts and origins differ in colony morphology, pathogenicity and growth rate, as well as conidial state. It has therefore been suggested that distinct strains, linked to host or geographical origin, are encompassed in C. fimbriata. The aim of this study was to develop PCR‐based microsatellite markers for population studies on C. fimbriata. ISSR‐PCR was used to target specific microsatellite regions of DNA from C. fimbriata. These amplified products were cloned and sequenced. Primer pairs were designed from these sequences to flank the microsatellite regions. From 24 primer pairs, 11 polymorphic primers were selected and tested on a number of C. fimbriata isolates representing a wide host and geographical range. Cluster analyses of the results indicate that these markers clearly distinguish between different geographical and host specific populations of C. fimbriata. The results are concordant with sequence data from the internal transcribed spacer (ITS) region of the rDNA operon of the same isolates. These markers will be useful in future studies of C. fimbriata population structure and diversity.