Background Daclatasvir (BMS-790052) is an investigational molecule that inhibits the HCV NS5A protein and shows potent antiviral activity apparently across all HCV genotypes. Selection of drug resistance mutations has been reported only for HCV genotype 1, and no information exists for other HCV variants and/or in HIV–HCV-coinfected individuals. Methods All interferon-α-naive, HIV–HCV-coinfected patients newly attended at Hospital Carlos III (Madrid, Spain) in 2011 were identified. Changes reported to be associated with daclatasvir resistance in the in vitro replication system for HCV genotype/subtypes 1a/1b (M28T, Q30H/R, L31F/M/V, P32L and Y93C/H/N) were examined. Results A total of 78 HIV–HCV-coinfected individuals as well as 635 NS5A sequences deposited at Los Alamos HCV database were analysed. None of the NS5A sequences from HCV-1a or HCV-3 showed changes associated with daclatasvir resistance. By contrast, all NS5A sequences from HCV-4 harboured L31M. The double mutant L31M+Y93H was found in 7% of HCV-1b and 13% of HCV-4. Finally, all NS5A sequences from HCV-1b and HCV-4 harboured changes at codon 28 (M28L) and 30 (L30R), which are of unknown significance. The rate of all these NS5A polymorphisms did not differ significantly when comparing HIV–HCV-coinfected patients and sequences from HCV- monoinfected subjects deposited at Los Alamos HCV database. Conclusions Primary resistance mutations to daclatasvir, an investigational HCV NS5A inhibitor, are not seen in HCV-1a or in HCV-3 as natural polymorphisms. By contrast, they can be recognized in most HCV-1b and HCV-4 strains, regardless HIV coinfection.