Gastric biopsy ssampling could affect accuracy of Helicobacter pylori clarithromycin resistance assessment due to coexistence of susceptible and resistant strains (i.e. heteroresistance) either in same gastric site (intraniche) or in two different gastric sites (interniche). This study aimed to assess differences in the H. pylori clarithromycin resistance prevalence in relation to the gastric biopsy sampling by using Taqman-real time polymerase chain reaction (PCR). The study enrolled 137 patients. Primary clarithromycin resistance was observed in 15 isolates exclusively in antrum, in 7 cases exclusively in gastric body, and in 3 patients in both gastric sites. The overall prevalence of clarithromycin resistance was 13.1% by using exclusively antral biopsies, and 18.2% by using biopsies from both gastric sites. Moreover, intra-niche heteroresistance was observed in 19 (76%) out of 25 patients harbouring resistant strains. Our data found a heterogeneous distribution of resistant H. pylori strains in the stomach. Similarly to culture, gastric biopsies from both antrum and gastric body are needed to increase the accuracy of PCR-based methods for clarithromycin resistance assessment.