Purpose: This study aimed to characterize common mutations of antibiotic-resistant gene of clarithromycin, metronidazole and levofloxacin in Helicobacter pylori (H. pylori) and determine their association with antibiotic resistance of H. pylori for providing a strategy for eradication therapy of H. pylori infection in children. Patients and Methods: The antibiotic resistance to clarithromycin, metronidazole and levofloxacin for H. pylori strains isolated from children was determined by E-test. The mutation of domain V of 23S rRNA, rdxA and frxA genes, gyrA and gyrB genes was performed by PCR-based sequencing of DNA fragments. Results: Out of the 79 H. pylori strains examined, 66 (83.5%) were resistant to at least one of the tested antibiotics and 13 (16.5%) were fully sensitive. A total of 29 (36.7%) strains were resistant to clarithromycin. Analysis of the 23S rRNA gene showed that most mutations occurred at the A2143G and T2182C sites, showing a frequency of 82.8% (24/29) and 89.7% (26/29) respectively. In the 11 sensitive strains to clarithromycin, the frequency of A2143G mutation was only 45.5%, which was significantly lower than that in resistant strains (P<0.05). There were 54 strains (68.4%) resistant to metronidazole, with most mutations occurring at G47A and T184G in the rdxA gene. T184G mutation was recognized in metronidazole-sensitive strains, but no G47A mutation was identified. Twelve strains (15.2%) were resistant to levofloxacin. Position 91 mutation of the gyrA gene occurred only in resistant strains, whereas position 87 mutations were detected in both sensitive and resistant strains to levofloxacin. Conclusion: In H. pylori resistant strains isolated from children in China, most mutations occurred at A2143G of the 23S rRNA gene for clarithromycin; G47A mutation of rdxA gene for metronidazole; and at 91 mutation of gyrA gene for levofloxacin. It is suggested that susceptibility testing together with screening the mutation of antimicrobial-resistant gene prior to treatment is important for the eradication of H. pylori in children.