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Abstract Background Blepharis constitutes an important part of the vegetation of the Jordanian arid and semi-arid regions, yet whether one or more species of this genus occurs in the Jordanian area is uncertain. We addressed this question by assessing morphological characters and testing Inter-Simple Sequence Repeat (ISSR) markers from 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 randomly chosen Blepharis plants were harvested from each of the three populations for morphological and molecular analyses. In the northern populations, spikes were lax and bract width was significantly shorter than length of the longest lateral spine compared to the southern population. A multivariate linear discriminant analysis distinguished the northern populations from the southern one by internode length, bract width, longest lateral spine length, and bract width to spine length ratio. 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 and corresponding with the “northern” and “southern” population designations. Conclusions Our results strongly indicate the occurrence of two discrete Blepharis species in Jordan and reject the hypothesis that the genus is represented by only one species. We propose that the Blepharis 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 and revising floristic work within the region and an updated key has been included in our findings.