Introduction: Tobacco, scientifically known as Nicotiana tabacum L. is an annual allotetraploid plant (2n=4x=48) in the Solanaceae family and is widely used as a model in plant biology. Oriental tobacco is a sun-cured, highly aromatic, small-leafed type which is mainly grown in Turkey, Iran, Greece, Bulgaria, Lebanon and the Republic of Macedonia. It is capable of growing in low fertile soils. To create an American Blend cigarette, it is necessary to mixed oriental tobacco with more robust tobacco such as Virginia and Burley tobaccos. Estimating genetic diversity and knowledge on genetic relationships among genotypes is a crucial aspect of promoting and implementing breeding programs,without it, effective and desirable genetic modification cannot be accomplished. Various types of marker including morphological, bioCHEMICAL, and molecular ones are used by plant breeders to estimate genetic diversity. The purpose of this study is to investigate the genetic diversity in the population of recombinant inbred lines for CHEMICAL characteristics such as chlorine accumulation in leaves, nicotine, sugar and ash. Materials and Methods: In the present study, genetic diversity of an oriental tobacco population consists of 55 recombinant inbred lines,coming from Basma seres 31 × SPT 406 cross, was assessed for CHEMICAL TRAITS in randomized complete block design with three replications. From the middle leaves of each genotype, 20 leaves were randomly selected in each replication and the percentage of CHEMICAL elements such as chlorine, sugar content, nicotine contentand ash content was determined using the method proposed by CORESTA (Cooperation Center for Scientific Research Relative to Tobacco). The identification of outlier data and the test of normality of the distribution of experimental errors were performed according to Shapiro and Wilke's method in SAS software version 4. 9. Analysis of variance of the CHEMICAL TRAITS data was performed according to the statistical model of randomized complete block design in the SAS software. After standardizing the data, the cluster analysis was carried out using the minimum variance method. Principal components analysis was performed using the correlation coefficients matrix of TRAITS. The average comparisons of the groups resulting from the cluster analysis was done in SPSS version 20 software using the SNK method. Results and Discussion: Analysis of variance revealed significant difference among the studied lines for nicotine and chlorine concentration. There was significant negative correlation between nicotine and sugar and significant positive correlation was observed between sugar and ash. Principal component analysis revealed 3 of the 4 main components play the most important role in explaining the total diversity among individuals and all TRAITS except chlorine concentration showed negative correlation with the first component. Using cluster analysis by Ward’, s method, the studied population was grouped into 4 separate subgroups. Groups mean comparisons using SNK test showed that individuals in group 1 have the higher values for most of the studied TRAITS. Conclusion: According to the present study, a wide genetic diversity was observed for nicotine and chlorine TRAITS in the studied population, which can be used in breeding programs for oriental tobacco. A negative correlation was observed between nicotine and sugar, and a positive correlation between sugar and ash. This can be used to modify correlated attributes, as increasing or decreasing the value of a trait increase or decrease the TRAITS associated with that trait. Individuals of the studied population were divided into 4 separate groups, which can help researchers in choosing parents because offspring resulting from the crossing of distant parents,show more heterosis and diversity. Production of desirable varieties increases the farmers' incomes by reducing the average production costs.