We investigated potential patient-to-patient transmission of hepatitis C virus (HCV) in two hemodialysis centers in Beijing, China. Approximately 8.25% (32/388) hemodialysis patients were HCV antibody positive, and 4.90% (19/388) were HCV RNA-positive, which consisted of 2a genotype (1/19) and 1b genotypes (18/19). Using next generation sequencing (NGS) approach, MiSeq platform, we sequenced HCV, targeting hypervariable region 1 (263 base-pairs) of genotype 1b specimens and obtained 18 to 243 unique HCV variants. Analysis of phylogenetic tree, viral epidemiology signature pattern (VESP) and Shannon entropy indicated no obvious HCV similarity for most HCV infections but limited HCV variants from Patient 31 (P31) were closer with respect to evolutionary relationship with Patient 24 (P24). However, it was unlikely that HCV was transmitted directly from P24 to P31 in the hemodialysis center. Otherwise, their genetic distance (3.92%-8.92%), would have been much less. Moreover, P31 was infected less than two years before specimen collection, and other external high risk factors existed for these two patients. Thus, our data indicated no evidence of patient-to-patient transmission of HCV in the two hemodialysis centers, suggesting that current HCV infection control measures are effective.