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Artemisia herba-alba is an herbaceous aromatic and therapeutic plant widely distributed in semi-arid regions of Tunisia and is potentially usable to restore degraded ecosystems. A study of genetic variation among 216 accessions was conducted using ISSR (Inter Simple Sequence Repeat) markers to assess the polymorphism at the species level. A total of 60 polymorphic loci were scored using four primers revealing a high level of genetic polymorphism among A. herba-alba accessions. Correlation analysis revealed no direct relation between morphological traits, geographic distance and genetic distance. Correlogram analysis showed a patchy distribution of the genetic variability of A. herba-alba accessions revealing the contribution of local ecological and geographic conditions on variability.