Objectives: to determine the frequency of viral genotypes and subtypes of hepatitis C virus (HCV) in Colombian patients observing possible associations with variables such as age, sex and viral load. Methodology: we performed genotyping and subtype of HCV in chronically infected patients treated at different sites of the clinical laboratory of a third level health care service providing entity present in different cities in Colombia using the HCV TRUGENE 5NC system. The system consists of the sequencing of the cDNA obtained from the virus by RT-PCR from each sample of patients included in the study. Results: we performed HCV genotype analysis in 284 patients treated in different cities during the years 2008, 2009 and first quarter of 2010. It was possible to detect genotypes 1, 2, 3 and 4 of hepatitis C virus subtypes and 1 a, 1b, 2a, 2b, 3a y 4c, with the following frequencies 16.20%, 71.13%, 2.11%, 2.11%, 0.35%, 0.35%, respectively. Mixed infection by subtypes 1a and 1b were detected in 7.8% of the cases. Women represented a high proportion of the cases included in the study (56.7%), while 45 - 64 years was the age range mostly represented (83.1%) in the sample analyzed. HCV viral load was usually higher in men (p = 0.004); and as a trend, men presenting subtype 1a also showed the highest viral loads. Conclusions: we showed a higher prevalence of genotype 1, observed in 95% of the cases analyzed. Among these cases, viral subtype 1b was the most frequently found in the population analyzed (71.13%).