Due to the similar routes of transmission, individuals infected with the human immunodeficiency virus (HIV) may become infected with the hepatitis C virus (HCV) simultaneously. The aim of this study was to investigate the frequency of HCV co-infection in Iranian individuals with HIV infection, and to genotype HCV in plasma and PBMC specimens of these patients. From September 2015 to October 2016, a total of 140 Iranian individuals with HIV infection were enrolled in this cross-sectional study. The RNA from plasma and PBMC specimens was extracted, and genomic HCV-RNA was amplified using RT-nested PCR with primers that target 5'-UTR. The HCV genotyping used the RFLP technique. To confirm HCV genotype, 10 randomly selected HCV-positive samples were also submitted for sequencing. The mean age of patients was 35.7 ± 13.5 years (range: 1-66). Out of 140 patients, 62 (44.3 %) were positive for anti-HCV antibodies; among these, viral genomic RNA was detected in 34 (24.3%) and 39 (27.9%) of the plasma and PBMC specimens, respectively. The HCV genotyping showed a similar pattern of subtypes 1a (44% vs 46.2%), 3a (32.4% vs 33.3%), and 1b (17.6% vs 17.9%) in all sera and PBMC samples. It is noteworthy that the HCV genotypes in plasma and PBMC specimens of 6 HCV co-infected patients were not the same. This study reveals that HIV/HCV co-infection is high in Iranian patients (44.3%), especially in people who have high-risk factors (83.9%). Also, HIV/HCV co-infected individuals may have dissimilar HCV genotypes in their plasma and PBMC specimens.