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Fusarium pseudograminearum is one of the major pathogens causing crown rot of wheat in the semi-arid and arid areas in Tunisia. In this study, the molecular diversity of 74 isolates of F. pseudograminearum representing three populations from Tunisia and a set of isolates from the world collection was investigated. The potential mycotoxin-producing ability was tested by PCR using primer pairs specific for the Tri3, Tri7 and Tri13 genes. Results indicated that all the isolates are potentially DON and/or 3-AcDON producers. The mating-type idiomorphs were identified using diagnostic PCR primer for MAT1-1 and MAT1-2. Both mating types were recovered from the same region and in some cases from the same field. Restriction analysis of the nuclear ribosomal DNA (nrDNA) intergenic spacer region (IGS) revealed 11 haplotypes, five of which were identified in the world collection. The analysis of population structure using the combined IGS and MAT data revealed that the total gene diversity (H(T) = 0.108) was mostly attributable to diversity within populations (H(S) = 0.102) and that the genetic differentiation among the four populations was low (G(ST) = 0.09). The analysis of molecular variance (amova) showed that 15% of the variability was between the Tunisian populations and the world collection. These findings indicate that quarantine measures should be in place to limit the introduction of new populations of F. pseudograminearum into Tunisia.