Hepatitis C virus (HCV) infection is one of the leading causes of chronic liver disease and has emerged as a global concern of public health, affecting about 3% of the world’s population. Pakistan is the sixth most populated country in the world and has a HCV prevalence rate of 5.9%. While there are different subtypes of HCV, genotype 3a is the most common form in patients from Pakistan. The clinical outcome of HVC infection is determined by the interplay between viral, environmental and host related factors. The host’s immune system is the most important factor in viral persistence and innate immunity is the first line of defense, intervening with interferons and natural killer cells. On the basis of the available information regarding the HCV patient’s treatment with pegylated interferon-α and ribavirin, it has been observed that less than 50 % patients show the sustained virologic response six months after treatment cessation. Genome wide association studies have been linked to the responses against the treatment. Therefore this study was designed to identify the variations in the IL 28-B in the HCV patients which does not show the sustained virologic response against the pegylated interferon- α and ribavirin therapy. The IL28 B haplotypes associated with treatment response are also associated with spontaneous HCV clearance. IL28 B and racial differences in HCV control is because of SNPs in IL28B gene, 3’ un-translated region and promoter region and these have varied distributions among ethnic groups. It is fascinating to consider whether the differences at this locus account for the association between different ethnic groups and increased spontaneous and treatment induced clearance. In this study we selected HCV positive patients including male and female with different age groups. The viral loads were quantified on real-time PCR at base line and after 24 weeks of treatment of patients with pegylated interferon- α and ribavirin therapy. Hepatitis C virus genotyping was performed to classify the virus in different genotypes. The role of the genetic polymorphisms present in interferon-λ genes was investigated by scanning an area of 624 Kb on chromosome 19q13.13 (nucleotide positions, 39719200 – 39781600) encoding IL 29 (IFNL1), IL28A (IFNL 2), IL28B (IFNL3) and IFNL 4. Out of fifty SNPs, twenty five were designed in the IL28B gene coding region, which is considered to be the main factor for inducing the expression of Interferon stimulated genes (ISGs) using the iPLEX assay on a SEQUENOM MassARRAY® platform. Twenty three SNPs covered the 3’ and 5’ UTR’s of all four IFN-λ genes. The remaining two SNPs were designed in the newly discovered IFNL-4 gene. Our findings support that younger HCV patient’s shows better response to interferon and ribavirin combination therapy than older ones. Females aged ≤ 30years is a favorable marker. HCV genotypes showed different geographic distribution in regions of Punjab province. Over all distribution of HCV genotype in Punjab were given as 3a= 81%, 1a= 11%, 4a= 4%, 3a+1a=3%, and 1a+4a =1 %. It is further evident from the findings of the present study that in Pakistan more than one genotype exists. HCV genotypes 1 and 4 have been detected with genotype 3a being most frequent, which predict short duration with better biochemical and virologic response. Therefore, it is beneficial to include the predictive variables such as genotype in the cost-benefit analysis of HCV treatment to plan policies to increase the effectuality. These results can be used to counsel patients on the likelihood of response and may influence the patient’s decision on treatment. The allelic association data revealed that a region of ~ 39 Kb (Chr. 19, nucleotide positions, 39729450-39768250; build GRCh37.p10) containing 13 polymorphic SNPs in Pakistani population is strongly associated (Fisher’s P value = 0.0003-0.0130) with spontaneous clearance and for all of these SNPs, spontaneous HCV clearance was more common with the major alleles. The most significant results were obtained with RS8109886 (Odds ratio of presenting HCV clearance [OR] for C vs. A = 3.6 [95% CI: 1.9-6.5] Fisher’s P = 0.0001), RS8113007 (A vs. T OR = 3.6 [1.9-6.5]; Fisher’s P = 0.0001) and RS12979860 (C vs. T OR = 3.1 [1.7-5.3]; Fisher’s P = 0.0002). The linkage disequilibrium data performed between 23 polymorphic IFN-λ region SNPs, revealed three haplotypic blocks: haplotype block I, of eight Kb, included eight SNPs (RS35790907, RS12972991, RS12980275, RS12982533, RS8105790, RS688187, RS4803217 and RS12979860) in strong linkage disequilibrium (r2 ≥ 0.85) among themselves and haplotype, haplotype block II, of 4Kb included seven SNPs (RS4803221, RS1549928, RS10853727, RS109886, RS8113007, RS8099917, RS7248668) in strong linkage disequilibrium (r2 ≥ 0.95) among themselves and haplotype and block III contained just two SNPs (RS1671087 and RS11665818) lying approximately 6 kb apart from each other and in strong linkage disequilibrium (r2 ≥ 0.85%). In total 6 haplotypes were investigated comprising of 15 IFN-λ region SNPs, haplotype one (AAATTGCCCATCATG) comprising of major alleles of 15 SNPs (RS35790907, RS12972991, RS12980275, RS12982533, RS8105790, RS688187, RS4803217, RS12979860, RS4803221, RS1549928, RS10853727, RS8109886, RS8113007, RS8099917 and RS7248668) had most significant association (OR = 2.37, 95% CI = 1.34-4.20, P = 2.8x10-3) with therapy response in comparison with other detected haplotypes. HCV clearance is a complex process that is dependent on the type of HCV infection and the host’s immunity-related genetic factors. Some SNPs that are associated with HCV clearance in Pakistani patients are the same as those that have been associated in other cohorts and suggest a common genetic background across multiple populations for HCV clearance. However, a number of alleles identified in this study were unique to the patient cohort and could suggest Pakistani-specific factors for HCV clearance, particularly for genotype 3a. These data, however, do support findings that have been widely reported from other groups where host genotype has been a proven factor in HCV clearance and treatment response. Tailoring treatments to target potential responders, as opposed to generalized, universal treatment strategies, will be of economic benefit but more importantly will have substantial patient benefits as patients would recover quicker and be less likely to require multiple ‘trial-and-error’ treatments. Our data supports the associations of SNPs present in the IFN-λ genes with HCV clearance after interferon and ribavirin combined therapy in Pakistani individuals infected with genotype 3a and provides preliminary evidence to suggest patients should be genotyped for the relevant SNPs in order to predict drug response before starting therapy.