Objective To compare the composition and diversity of lung and gut microbiota between Chinese and golden hamsters using 16S rDNA sequencing, to provide a new perspective for understanding their applications in human disease research. Methods SPF-grade Chinese hamsters and golden hamsters (6 males and 6 females per group) aged 6 ~ 8 weeks were selected. Bronchoalveolar lavage fluid, small intestinal contents, and large intestinal contents were collected under sterile conditions. Species annotation and α-diversity and β-diversity analyses were performed using 16S rDNA sequencing. Linear discriminant analysis effect size ( LEfSe) analysis was employed to identify potential biomarkers. Results The α-diversity of the lung and small intestinal microbiota was significantly higher in golden hamsters (especially males) than in Chinese hamsters (P<0. 05), while the diversity of the large intestinal microbiota was significantly higher in Chinese hamsters ( P<0. 05 ). Regarding β-diversity, the composition of the lung, small intestinal, and large intestinal microbiota differed significantly between the two types of hamsters (P<0. 01). LEfSe analysis revealed differential occurrence of the phylum Firmicutes in the lung microbiota in golden hamsters, and differential occurrence of the phyla Bacteroidota ( females) and Proteobacteria (males) in Chinese hamsters. Regarding the small intestinal and large intestinal microbiota, Firmicutes and Bacteroidota were differential phyla in Chinese and golden hamsters, respectively. Conclusions The composition and diversity of the lung and gut microbiota differed significantly between Chinese and golden hamsters, which may be related to their distinct applications in human disease research. This study provides a new perspective for understanding the applications of these two types of hamsters in human disease research and lays a theoretical foundation for future efforts to improve their health status and their value as experimental models by modulating their microbiota.