Current management of HCV infection is based on Direct-Acting Antiviral Drugs (DAAs). However, resistance-associated mutations, especially in the NS3 and NS5B regions are gradually decreasing the efficacy of DAAs. The aim of the current study was to identify such mutations in the NS3, and NS5B genes in DAAs treatment-naïve Pakistani chronic HCV 3a patients. Peripheral blood samples were collected from 233 chronic HCV 3a patients at different tertiary care hospitals in Karachi, Pakistan, between August 2020 to September 2021. PCR-amplified target regions of the NS3/NS5B gene were subjected to Sanger sequencing to identify resistance-associated mutations. Phylogenetic analysis of the identified amino acid sequences was performed using HCV3a sequences of the global population in the virus pathogen resource (VIPR) database. Sequence analysis identified five amino acid mutations, Leu36Pro, Gln41His, Gln80Lys/Arg, Ala156Tyr, and Gln168Arg in the NS3 region, and two mutations Leu159Phe and Cys316Arg in the NS5B region. Phylogenetic analysis revealed a high genetic diversity in the studied isolates. Overall, the prevalence of resistance-associated substitutions was almost similar to other geographic regions worldwide. This data could be helpful in selecting the most effective treatment regimen for HCV chronically infected people in Pakistan.