Hepatitis C Virus (HCV) infection is one of the leading risk factors for end-stage liver disease development worldwide. This RNA virus displays high genetic diversity with 8 genotypes and 96 subgenotypes with heterogenous geographical distribution around the world.In this study we carried out an active case finding of individuals with history of transfusion events before 1996 in three cities in Colombia. Then, the characterization the HCV genotypes, subgenotypes, and resistance associate substitutions (RAS) was performed in samples positives for antibodies anti-HCV + from this study population. In addition, samples from PWID and patients with end-stage liver disease and submitted to liver transplantation were included in the phylogenetic and RAS analysis.The 5'UTR, NS5A, and NS5B regions of the HCV genome were amplified in serum or liver explants samples. After edition, assembly and alignment of the sequences, genotyping through phylogenetic analysis was performed using IQTREE V2.0.5 based on maximum likelihood approach. The identification of RAS was carried out by alignments based on the reference sequence (GenBank NC_004102).Two hundred sixty individuals with blood transfusion events before 1996 were recruited. The seroprevalence of antibodies anti-HCV was 2,69% in this population. The HCV genotypes 1, 2 and 4 and subgenotypes 1a, 1b, 2a, 4a and 4d were characterized in the study populations. Three RAS (Q30R, C316N and Y93H) were identified in samples obtained from 2 individuals who received blood transfusion before 1996 and without previous antiviral treatment and 6 samples obtained from patients end-stage liver disease.The HCV genotype 1, subgenotype 1b, is still the most frequent in this middle-income country, regardless the risk factor. We report the first characterization of HCV subgenotypes 4a and 4d and the first RAS identification in patients in Colombia.