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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 …