Genetic variability and population structure were studied in 11 geographical populations of Lallemantia royleana Benth. (Lamiaceae). DNA was extracted from 102 plant specimens and used for ISSR assay. Genetic diversity parameters were determined in these populations. AMOVA and Gst analyses revealed the presence of extensive genetic variability within populations and significant molecular difference among the studied populations. Mantel test showed positive significant correlation between genetic distance and geographical distance of the populations. STRUCTURE and K-means clustering revealed populations genetic stratification. Networking showed some degree of gene exchange among the studied populations. UPGMA dendrogram of the populations based on morphological characters was in agreement with the NJ tree of molecular data. These results indicated that geographical populations of Lallemantia royleana are well differentiated both in genetic content and morphological characteristics. This information may be of use for future conservation and breeding of this medicinally important plant species.