Abstract: This study describes the application of the EcoTILLING method, using the denaturing High-Performance Liquid Chromatography (DHPLC) system, for the detection of a polymorphic region in the chloroplast gene ribulose 1, 5-bisphosphate carboxylase gene (rbcL) of Eryngium spp. Five Eryngium L. species: E. maritimum, E. creticum, E. glomeratum, E. campestre, E. billardieri and two ecotypes of E. creticum were collected from several locations in Syria and used in this study. Chromatographic data, confirmed by sequencing results, showed the presence of two SNPs in E. glomeratum (C/T and A/G) and one SNP in each of E. campestre, E. creticum eco. Slenfeh and E. creticum eco. Dara'a (A/G). The detected polymorphic region was tested for a successful separation of plant families and sub families. The procedure described in this paper enables low cost, highly sensitive and high-throughput sample screening which enables the assembly of information on candidate polymorphic regions in genes of interest to phylogenetics.