The development of direct-acting antivirals (DAAs) has significantly improved hepatitis C virus (HCV) treatment. However, drug resistance remains a potential concern in the real-world DAA-based therapies. We previously developed a novel full-length genotype 2a HCVcc clone PR63cc directly from clinical isolates. Here in this study, we compared the sensitivity of PR63cc and JFH1 to 12 different DAAs most of which are either already in clinical use or in the late clinical development phase. For NS5B inhibitors, PR63cc and JFH1 displayed comparable sensitivity to nucleoside/nucleotide analogues sofosbuvir and 2′-C-methyladenosine, while PR63cc was 4-fold more sensitive than JFH1 to nesbuvir, a non-nucleoside inhibitor. Interestingly, PR63cc and JFH1 were both completely resistant to dasabuvir which efficiently inhibited the replication of genotype 1b HCV replicon. For NS5A inhibitors, while PR63cc was as sensitive as JFH1 to ombitasvir and velpatasvir, it was much more resistant than JFH1 to daclatasvir and ledipasvir, which was mainly due to methionine at amino acid residue 31 of NS5A. For NS3 inhibitors, PR63cc was generally less sensitive than JFH1 to simeprevir, grazoprevir, asunaprevir and paritaprevir. Serine at residue 67 of NS3 was identified to be a resistance-associated variant (RAV) for asunaprevir. Finally, we showed that PR63cc was more resistant than JFH1 to the asunaprevir/daclatasvir combination treatment. In summary, our study systemically analyzed the DAA sensitivity of a new HCVcc strain and identified critical RAVs. These results are not only important for monitoring the emergence of drug-resistant mutations of current DAA therapies, but also valuable for developing next-generation DAAs.