【Abstract】 Objective This study aims to compare the composition and diversity of lung and gut microbiota between Chinese hamsters and golden hamsters using 16S rDNA sequencing technology, providing a new perspective for understanding their applications in human disease research. Methods Twelve SPF-grade Chinese hamsters and twelve golden hamsters (six males and six females each) aged 6 to 8 weeks were selected. Bronchoalveolar lavage fluid, small intestinal contents, and large intestinal contents were collected under sterile conditions. Species annotation and diversity analysis, including α-diversity and β-diversity analyses, were performed using 16S rDNA sequencing technology. Additionally, LEfSe analysis was employed to identify potential biomarkers. Results In the α-diversity analysis, the diversity of the lung and small intestinal microbiota in golden hamsters (especially males) was significantly higher than that in Chinese hamsters (P < 0.05), while the diversity of the large intestinal microbiota in Chinese hamsters was significantly higher than that in golden hamsters (P < 0.05). In the β-diversity analysis, significant differences were observed in the composition of the lung, small intestinal, and large intestinal microbiota between the two types of hamsters (P < 0.01). LEfSe analysis revealed that in the lung microbiota, the differential species in golden hamsters were the phylum Firmicutes. In Chinese hamsters, the differential species were the phylum Bacteroidota (females) and the phylum Proteobacteria (males). In the small intestinal and large intestinal, the differential species in Chinese hamsters were the phylum Firmicutes, while in golden hamsters, the differential species were the phylum Bacteroidota. Conclusions Significant differences exist in the composition and diversity of lung and gut microbiota between Chinese hamsters and golden hamsters, which may be related to their distinct applications in human disease research. For example, the predominance of the phylum Firmicutes in Chinese hamsters may be associated with their characteristics as models for type 2 diabetes research, while the predominance of the phylum Bacteroidota in golden hamsters may influence their susceptibility to SARS-CoV-2. 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 experimental performance by modulating their microbiota.