Hepatitis C virus (HCV) infection affects ≥ 180 million individuals worldwide especially those living in developing countries. Recent advances in direct-acting therapeutics promise effective treatments for chronic HCV carriers, but only if the affected individuals are identified. Good treatment coverage therefore requires accurate epidemiological data on HCV infection. In 2014, we determined the current prevalence of HCV in Thailand to assess whether over the past decade the significant number of chronic carriers had changed. In total, 5964 serum samples from Thai residents between 6 months and 71 years of age were obtained from 7 provinces representing all 4 geographical regions of Thailand and screened for the anti-HCV antibody. Positive samples were further analyzed using RT-PCR, sequencing, and phylogenetic analysis to identify the prevailing HCV genotypes. We found that 56 (0.94%) samples tested positive for anti-HCV antibody (mean age = 36.6±17.6 years), while HCV RNA of the core and NS5B subgenomic regions was detected in 23 (41%) and 19 (34%) of the samples, respectively. The seropositive rates appeared to increase with age and peaked in individuals 41-50 years old. These results suggested that approximately 759,000 individuals are currently anti-HCV-positive and that 357,000 individuals have viremic HCV infection. These numbers represent a significant decline in the prevalence of HCV infection. Interestingly, the frequency of genotype 6 variants increased from 8.9% to 34.8%, while the prevalence of genotype 1b declined from 27% to 13%. These most recent comprehensive estimates of HCV burden in Thailand are valuable towards evidence-based treatment coverage for specific population groups, appropriate allocation of resources, and improvement in the national public health policy.