Exploitation of wheat germplasm requires the knowledge of genetic diversity of the plant materials. Althoughinheritanceof planttraitilants mainly depends upon nucleargenes, cytoplasmicfactors and their interactioalsolay important role. In this regard, the development and utilization of alloplasmic lines have facilitates the cytoplasm genetic studies. Due to the slow evolution of the chloroplastic genes, chloroplastic microsatellite loci, whose evolutionary speed is several times higher than other regions of the chloroplast genome,are suitable candidate for analysis of cytoplasmic variation. Therefore, 32 alloplasmic lines accompanied with a euplasmic parental line(control)were evaluated using morph physiological traitand molecular markers (microsatelite) during 2010-2011 and 2011-2012, using randomized completelock design. The results showed that 20 primer pairs out of 26 chloroplastic microsatellite markers (cR) are polymorphic. A total of 50 alleles were identified with an average of 2.5 alleles per locus. In this study, mean of polymorphism information content (PIC) was 0.33 and ranged from 0.05 (in WCt17 primer) to 0.49. (In WCt9 primer). After scoring electrophoresis bands from each primer pair using software Mega4, distance matrix based on p-distance was calculated to compare the two group of alloplasmic lines. Genetic distance between alloplasmic lines ranged from 0 to 0.76 with an average of 0.28. Cluster analysis of molecular data, revealed that used alloplasmic wheat lines belongs to two major clusters. Hence, differentiation of the cytoplasmic type and of the genus ( Triticum و Aegilops ) has been evident. Analysis of molecular variance (AMOVA) also showed significant differences between the two study groups. (F ST = 0.67 , P 0.001). Based on the analysis morpho-physical data, cytoplasm had no significant influence on chlorophyll content in leaves, dry weight, number of grain per spike, flag leaf length, peduncle length and spike length. On the other hand, height, photosynthesis, and seed yield, has been significantly affected by cytoplasm. It is concluded that hexaploid and tetraploid derived cytoplasms performed superior than diploid cytoplasm in wheat. However, The dendrogram obtained from morphological data failed to fully segregate the two genera. Results comparing the similarity of two clusters of molecular and morphological data, showed no significant relationship. This inconsistency can be justified based on hypothesis that amplification of cR markers specific for the repetitive regions of the chloroplast genome within non-coding regions while the coding regions of the genome is expressed via morphological markers. Overall, the results showed that the primers used can be valuable resources of polymorphic markers for the analysis of cytoplasm of Triticeae species, with the potential of clearly differentiate in close species and genera of this tribe.