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Seminal fluid, once believed to be sterile, is now recognized as constituting a complex and dynamic environment inhabited by a diverse community of micro-organisms. However, research on the seminal microbiota in chickens is limited, and microbiota variations among different chicken breeds remain largely unexplored. In this study, we collected semen samples from Beijing You Chicken (BYC) and Tibetan Chicken (TC) and explored the characteristics of the microbiota using 16S rRNA gene sequencing. Additionally, we collected cloacal samples from the TC to control for environmental contamination. The results revealed that the microbial communities in the semen were significantly different from those in the cloaca. Firmicutesand Actinobacteriotawere the predominant phyla in BYC and TC semen, respectively, with Lactobacillusand Phyllobacteriumbeing the dominant genera in each group. Additionally, the seminal microbiota of BYC exhibited greater richness and evenness than that of TC. Principal coordinate analysis (PCoA) indicated significant intergroup differences between the seminal microbiotas of BYC and TC. Subsequently, by combining linear discriminant analysis effect size and random forest analyses, we identified Lactobacillusas the predominant microorganism in BYC semen, whereas Phyllobacteriumdominated in TC semen. Furthermore, co-occurrence network analysis revealed a more intricate network in the BYC group than in the TC group. Additionally, unique microbial functional characteristics were observed in each breed, with TC exhibiting metabolic features potentially associated with their ability to adapt to high-altitude environments. The results of this study emphasized the unique microbiota present in chicken semen, which may be influenced by genetics and evolutionary history. Significant variations were observed between low-altitude and high-altitude breeds, highlighting the breed-specific implications of the seminal microbiota for reproduction and high-altitude adaptation.