Sixteen Tunisian natural populations of Hypericum humifusum from different bioclimates (sub‐humid, upper semi‐arid and lower semi‐arid) were assessed for their genetic diversity using nine isozymes. For all populations and all enzymes, 11 polymorphic loci were detected. The allelic frequencies varied according to the populations. A high genetic diversity and an excess of heterozygosity were observed within populations (A p = 2.02; P% = 64.29; H o = 0.321). The outbreeding mating system and the high number of individuals in the starting populations may contribute to this high variation. A relatively high level of differentiation (F ST = 0.142) and a restricted gene flow among populations (N m =1.34) were revealed. The lower semi‐arid populations showed the highest differentiation (F ST = 0.205). The relationship between F ST and geographic distance matrices was not significant, indicating that the genetic structure among populations is more closely linked to habitat fragmentation and probably to founder events. Nei’s unbiased genetic distances among pairs of populations were low (0.012–0.119). The unweighted pair group method with arithmetic averaging algorithm cluster based on these distances showed four sub‐clusters. Population groupings had no evident relationship with bioclimates or geographic regions. The substantial differentiation between populations and their high genetic similarity suggest their recent divergence as a result of habitat fragmentation. Most of the total variation was found within populations, thus ex‐situ conservation should be based on sampling seeds within, rather than among, populations. Populations harbouring rare alleles and a high genetic diversity level should be collected first.