Background: Blepharis constitutes an important vegetative part of the Jordanian arid and semi-arid regions, yet whether one or more species occurs in this area is debatable. We addressed this question by assessing morphological characters and employing Inter-Simple Sequence Repeat (ISSR) markers on three populations of Blepharis: two Northern (lower slopes of Kufranjah valley and the Dead Sea region) and one Southern (Wadi al Yutm). Results: Shoots from 19 randomly chosen Blepharis plants per population were harvested for morphological and molecular analyses. Spikes were lax and bract width was considerably shorter than length of the longest lateral spine in the Northern populations compared to the Southern population (P < 0.001). A multivariate linear discriminant analysis (LDA) distinguished the Northern populations from the Southern one largely by internode length, bract width, longest lateral spine length, and bract width to spine length ratio (P < 0.001). The ISSR analysis revealed that 44 markers across eight primers were polymorphic with major allele frequency of 83.6% and an average of 5.5 polymorphic markers per primer. The genetic resemblance among individuals ranged from 0.27 to 0.96. The three Blepharis populations were accordingly clustered into two distinct groups, similar to the analysis of morphological differences. Conclusions: Our results strongly indicate the occurrence of two discrete Blepharis species in Jordan and the hypothesis that the genus is represented by only one species is not supported. The species in Jordan are B. attenutata Napper (represented by the Northern populations) and B. ciliaris [L.] B. L. Burtt (represented by the Southern population). These findings are important for informing floristic work within the region and an updated key has been included in our findings.