The genetic structure of M. destructor and M. hordei was investigated by sampling 21 fields of cereals in 14 localities of central and southern Tunisia. As previously shown, there was no strict association between the cereal species (wheat, barley and oat) and the Mayetiola species. M destructor males displayed no heterozygosity at the Pgm3 locus, indicating that they were hemizygous as is the PGM locus in North America. In M. hordei, heterozygous males were observed at all loci, but strong heterozygote deficits were found at two loci (Mdh2 et Hk). Since no such deficit was observed in females, the population structure of M. hordei was studied only in females. Although heterozygosity was two fold higher in M. hordei than in M. destructor, the two species were similar for other genetic characteristics, including a low (Fst < 0.05) but significant (P < 0.05) genetic differentiation, no isolation by distance, and similar rates of gene flow (5.7 less than or equal to Nm less than or equal to 9.6). These results are discussed in relation to their consequences in the event of controlling Tunisian Hessian flies using wheat cultivars that are resistant to a M. destructor biotype.