The causal agent of Septoria tritici blotch, Mycosphaerella graminicola, is currently the most frequently reported pathogen on wheat crops worldwide. The populations of this fungus were previously studied in Tunisia using microsatellite markers, but no information is available on the mitochondrial (mt) genetic structure of the fungus in this country. The objective of this study was thus to examine the genetic diversity and population structure of M. graminicola in Tunisia using mtDNA markers. A set 105 monoconidial isolates of the pathogen were sampled in 2012 from four geographical locations in Tunisia (Bizerte, Béja, Kef and Jendouba) and assessed for diversity and polymorphism of three mtDNA sequences (Mg1: 388 bp, Mg2: 570 bp, Mg3: 929 bp) using PCR-RFLP-SSCP. Mg2 and Mg3 were the more polymorphic sequences with 12 and 9 observed alleles, respectively, while Mg1 was the less polymorphic one with only two alleles. Data analyses revealed a moderate mt gene diversity and a low mt haplotype diversity within the population. Further analyses showed a low population differentiation (G(ST) = 0.16) and a moderate gene flow (Nm = 2.61) within the population. Both UPGMA and Bayesian clustering methods confirmed this lack of mt genetic structuration. Our study allowed us to characterize for the first time mt genetic structure of M. graminicola in Tunisia and to report a moderate mt gene diversity, a low mt genotype diversity and a lack of mt genetic structure of the parasite in this country.