We investigated mtDNA genetic differentiation and the phylogenetic relationships of 11 populations of the oniscidean genus Ligidium. We studied nine populations from Greece, assigned to three nominal species (L. euboicum, L. germanicum and L. beieri), and two from central Europe (L. germanicum and L. hypnorum), applying RFLP analysis of two mtDNA segments (12S rDNA and 16S rDNA) ampliﬁed by PCR. The analysis revealed seven monomorphic and four polymorphic populations, and 17 different haplotypes, each one present in one population only. Several restriction enzymes produced population speciﬁc patterns, enabling discrimination among the different populations. The estimated net nucleotide divergence between populations ranges from 1.06 to 16.52%, while the high NST value (0.96) indicates the existence of increased interpopulation genetic differentiation that can be attributed to the extreme isolation among Ligidium populations. The relationships between haplotypes and populations are not congruent with their geographical distribution and the established taxonomy within the genus.