Seven populations of Holothuria polii were sampled from the eastern and western Mediterranean coastal waters of Tunisia and screened electrophoretically for genetic variation at 11 allozyme loci. Six among the seven polymorphic loci were out of Hardy–Weinberg equilibrium (HWE) in at least one population. In the same way, the multilocus test showed deviation from HWE in all populations. These populations showed heterozygote deficiency. Genetic variability was relatively low. The number of alleles per locus ranged from 2.09 to 2.27 (average = 2.15), and the observed heterozygosity varied between 0.14 and 0.20 (average = 0.17). The observed overall differentiation among populations was slight but significant, with a mean FST value of 0.024 (P < 0.001). Pairwise FST values reflected the differentiation of the two populations, which were at the margins of the range sampled, from all the others. Our data suggest a population structure consistent with separation by Mediterranean Sea basins that might reflect different local biogeographical zones.