Open AccessAegilops has been considered a complex genus with as many as 22 species in Syria. The current study has used 585 nucleotides from 5.8S nuclear ribosomal DNA gene and internal transcribed spacer 2 for these different species. These data were aligned manually and subjected to bioinformatics manipulation in order to construct the genetic relationship among these species. Three statistical methods (maximum-parsimony-MP, maximum-likelihood-ML and neighborjoining-NJ) were used to execute the most likely relationship. The constructed genetic relationship showed homogeneinty in clustering of the species of the same plant type (A, B or C) with each other. A single NJ tree and a single ML tree were obtained with slight difference in topology within each plant type. Both trees disagreed with our previous finding in that A. searsii, speltoides and A. longissima clustered in one group and the first two species were sisters while A. caudata was out. Therefore, A. speltoides was not the oldest among them and these differences could be related to the difference in taxon sampling size. This study, however, supported our previous molecular finding and did not support the previous karyotypic study in that A. searsii was not the oldest, A. caudata was not recently originated and both A. longissima and A. speltoides were not intermediate. The molecular markers and taxon sampling size are therefore mandatory in clarifying the genetic diversity of closely related species, particularly, those which possess an economic importance like Aegilops.