Background and Aims: Different hepatitis C virus (HCV) genotypes have characteristic geographical distribution. Identification of HCV genotype is an important factor in the progression, clinical outcome and therapy of HCV infection. The aim of this study was to determine the prevalence of HCV genotypes among HCV-RNA positive patients in Yazd, Iran. Materials and Methods: In this cross-sectional study, 150 HCV-infected individuals with detectable plasma HCV RNA levels were enrolled from January to August 2015. HCV-RNA was extracted from plasma samples and retro-transcribed to c-DNA. Then HCV genotypes 1, 2, 3a, 4 were determined using a PCR based genotyping kit. Results: A total of 150 HCV-positive patients with mean age 40.45±11.83 were enrolled in the study. 89.3% of participant were males and 10.7% were females. The most common genotype was 3a (52%), followed by 1a (28%). Mixed-genotype infection was 20% and the most prevalent mixed genotype was 3a/1a (83% of mixed genotypes). The other genotypes were 1a/1b/3a in 10%, 3a/2 and 1a/2/3a both in 3% of patients with mixed HCV genotypes. Conclusions: Unlike other regions of Iran, Genotypes 3a was predominant in HCV-RNA positive patients in Yazd province. Also, HCV mixed-genotype infections were more common than previously estimated in other studies from different parts of the country.