<p>Cytological characteristics of agricultural plants are considered essential, and barley, as an important grain, is no exception. The present study was conducted on 114 wild barleys (<i>H. vulgare</i> subsp<i>. spontaneum</i>) genotypes collected from western provinces of Iran that were located in the Zagros basin. For each karyotype of the ploidy level, the total number of chromosomes, the number of basic chromosomes, the number of satellites, and the satellite chromosome, along with the karyotype formula, were determined. The results indicated that the studied genotypes are diploid, with a consistent base chromosome number of <i>x</i> = 7, and no variation in this number was observed among the genotypes. Genotypes were categorized into 4 groups based on the karyotypic formula. According to Stebbins' two-way table, the studied genotypes were classified into their respective symmetry classes. Within the studied Iranian genotypes, Classes 2B, 2A, and 1B were observed for the first time. Descriptive statistics revealed that CV_CI, CV_CL, A2, DRL, DI, AI, and CV indices had the highest coefficient of variation among the examined genotypes. A significant difference was found at the 1% level between genotypes in terms of all chromosomal traits (CL, LA, and SA) measured based on variance analysis. Correlation analyses revealed that chromosome length parameters were significantly associated with both symmetry and asymmetry related karyotypic indices, indicating structured chromosomal organization and pronounced intraspecific cytogenetic variation among the studied genotypes. Cluster analysis was performed using the studied traits and indices for genotypes by Ward's method, that the genotypes were categorized into 5 clusters.</p>