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In Tunisia, Arbutus unedo populations are severely destroyed due to deforestation and over-collecting. The species occurs in small scattered populations decreasing progressively in size. Yet, there is a lack of information’s regarding the amount of genetic diversity and its repartition for this resource. In this work, we analyzed the polymorphism of six isozymes in order to assess genetic variability and structuring of 15 natural populations prospected in three geographical areas. The analysis of the level and the distribution of the genetic diversity in this species might help in its conservation. Out of the 13 loci detected for all populations and isozymes analyzed, 4 loci were polymorphic. Allelic frequencies differed according to populations and particular alleles characterized ecological groups. A high genetic diversity within populations (Ap = 2.2; P = 63.33%) was observed. A relatively low level of differentiation (F-ST = 0.031) coupled with a high gene flow among populations (Nm = 6.81) were revealed. The differentiation of populations within the same bioclimate group was substantial and the upper semi-arid populations showed the highest differentiation (F-ST = 0.093). The relationship between F-ST and geographic distance matrices was not significant, indicating that the genetic structure among populations is not affected by geographic barriers but it is rather related to ecological factors such as altitudes and rainfall. Nei’s (1978) genetic distances among pairs of populations were low (0.001-0.030). The UPGMA cluster based on these distances showed three sub-clusters. Population groupings occur without evident relationship to bioclimates or geographic regions indicating that differentiation occurs at a local space scale. The species conservation strategies (in situ or ex situ) and the selection of genotypes should take into account the genetic diversity level within populations. (C) 2012 Elsevier B.V. All rights reserved.