Microsatellite markers were used to assess genetic diversity of Mycosphaerella graminicola at a micro geographical scale in Tunisia. Forty five (45) isolates were sampled and assessed using seven pairs of single-locus microsatellite primers not previously tested on populations of M. graminicola in Tunisia. Genetic diversity of the field population ranged from 0.403 to 0.555 with an average of 0.484. A high level of genetic diversity was found at a sharp scale throughout the pathogen population tested. Among 45 isolates sampled, 39 different multi locus genotypes (MLG) were identified. Cluster analysis (UPGMA) showed that 86% of the isolates tested were distinct. The high degree of DNA polymorphism, the large number of different molecular genotypes and the pattern of cluster analysis suggest that sexual ascospores and/or asexual spores of a highly mutable local population could have contributed to the genetic diversity of M. graminicola in Tunisia.