Objective: To explore the characteristics of gastric microbiota in children with and without (Helicobacter pylori, H. pylori) infection who had family history of H. pylori infection. Methods: Mucosal biopsy samples of the gastric corpus and gastric antrum were collected during the gastroscope. And the gastric mucosa flora’s information of the two groups of children were obtained after sample DNA extraction, PCR amplification of the 16S ribosomal DNA (rDNA) V3-V4 region, high-throughput sequencing and data processing. All the samples with family history of H. pylori infection were divided into two groups, the H. pylori infection group (n=18) and the H. pylori non-infection group (n=24). Then the α-, β-diversity and bacteria abundance of the gastric microbiota were compared between the H. pylori infection and non-infection groups at different taxonomic levels. The differential microbiota was found out by LEfSe analysis, and then the function of microbiota predicted using phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) method. Results: There was statistically significant difference in α-diversity (P<0.05) between the two groups, indicating that the H. pylori non-infection group had higher microbial richness than the H. pylori infection group. Moreover, the β-diversity was significantly different as well (P<0.05), which meant that the microbiota composition of the two groups was different. At the phyla level, Proteobacteria, Firmicutes, Bacteroides, Actinobacteria, and Fusobacteria were dominant in the two groups. At the genus level, Bacteroides, Prevotella, Streptococcus, and Neisseria, etc. were dominant in the H. pylori non-infected group. Meanwhile, Helicobacter and Haemophilus etc. were dominant in the H. pylori infected group. LEfSe analysis showed that the relative abundance of Bacteroides etc. at the genus level in the H. pylori non-infected group was significantly higher than that in the H. pylori infected group. Functional prediction showed that Bacteroides were positively correlated with amino acid and vitamin metabolism, mitogen-activated protein kinase (MAPK), mammalian target of rapamycin (mTOR) signaling pathway and ansamycin synthesis pathway. Conclusion: The gastric microbiota between H. pylori positive and H. pylori negative in children with family history of H. pylori infection is significant different. Some gastric microbiota, such as Bacteroides, may have a potential relationship with H. pylori infection in children.