BACKGROUND: Hepatitis C virus (HCV) resistance-associated substitutions (RASs) have a significant impact on the treatment of HCV with direct-acting antivirals (DAAs). However, limited research has been conducted, and no standardized methods for detecting RASs in mainland China. OBJECTIVES: To develop and apply a novel method for detecting HCV RASs in HCV RNA-positive patients in Linzhou, China. STUDY DESIGN: In total, 103 HCV RNA-positive serum specimens and epidemiological questionnaires were collected. A PCR method for detecting HCV RASs encompassing the NS3 to NS5B region was developed. RESULTS: Demographic analysis revealed a predominance of females (66/103, 64.1 %), with an average age of 70 years. Genotype 1b (GT1b) (17/103, 16.5 %) and GT2a (86/103, 83.5 %) were identified. The prevalence of RASs was higher (17/17, 100 %) in GT1b than in GT2a (7/86, 8 %). In GT1b, a higher frequency of RASs was observed in the NS5B region (17/17, 100 %) than in the NS3 (14/17, 82 %) and NS5A (10/17, 59 %) regions. C316N was the most prevalent, followed by S122G (71 %) and R30Q (35 %). CONCLUSIONS: We introduced an innovative approach for the detection of HCV RASs and provided a wealth of information on HCV RASs in Linzhou, China. The findings support the cautious selection of treatment regimens, potentially improving patient outcomes.