Hepatitis C is a worldwide problem which lead to the death of millions of people every year. In thalassemia patients, there is more risk of Hepatitis C infection due to multi transfusion in thalassemia patients. There is likelihood that the different Genotypes of HCV may be present in patients of thalassemia due to infection with Hepatitis C during blood transfusion. The main objective of this study was to rule out different types of HCV RNA Genotypes in the patients Beta-thalassemia and to rule out the causative mutation of the patients with Beta Thalassemia. A total of 100 clinically confirmed patients of Beta thalassemia were included in this study those were enrolled at Thalassemia clinic, Sheikh Zayed Hospital/ Medical College, Rahim Yar khan. The screening of blood samples were performed for the detection of anti-HCV antibodies by ICT method. 42 (42 %) samples were found positive and 58 (58 %) were negative for anti-HCV antibodies. The samples with HCV positive results were processed for HCV RNA amplification by Real Time Polymerase Chain Reaction (PCR) method. HCV-RNA was detected in 18 (7.56 %) samples while 24 (10.0 %) samples were HCV-RNA not detected. These Real time PCR assay was run for the characterization of HCV genotype and found that 17 out of 18 samples were of HCV RNA Genotype 3 while single RNA was The HCV RNA Genotype 1. Therefore, HCV RNA Genotype 3 is most predominant type of HCV in Rahim Yar Khan Region of the Punjab province. It can also be depicted that HCV Genotype 3 is the most wide spread strain in Thalassemia patients. There was no strong association of liver function tests was found in HCV positive patients. The DNA from these patients was investigated for screening of genetic variations by Sanger?s DNA sequencing method in selected region of Beta Hemoglobin gene (HBB) i.e. present on chromosome 11. The sequence analysis revealed a single base change (G>C) in intronic region at c.507 in 30% samples while a single nucleotide variation (T>C) at c.58 was found in 70% DNA sequences. The haplotype analysis was carried out and no significant impact of the variation was found on protein structure and function i.e. synonymous change of codon CAT>CAC (His=). So the identified genetic variations in the selected regions of Beta Hemoglobin gene (HBB) have no significant impact on its phenotype in Thalassemia patients from Rahim Yar Khan District of the Punjab, Pakistan.