Abstract: 【Objective】This study aimed to investigate the cecal microbiota community structure of three local chicken breeds in Guizhou based on microbial 16S rRNA sequencing, identifying specific microbiota enriched in the ceca of different chicken breeds, studying the characteristics of local chicken genetic resources from the perspective of intestinal microbota,thereby deepening the understanding of the characteristics of cecal microbiota in Guizhou local chickens.【Method】Three Guizhou chickens breeds(11 Wumeng black-bone chickens, 12 Qiandongnan Xiaoxiang chickens, and 12 Yaoshan chickens) were selected, and their cecal content samples were collected for 16S rRNA sequencing of the V3-V4 variable region. The QIIME software was used to calculate the alpha diversity of cecal microbiota, and principal coordinate analysis(PCoA) was conducted to evaluate cecal microbiota beta diversity. SparCC algorithm was employed to construct cecal microbiota interaction networks, and the Matthew's correlation coefficient(MCC) algorithm was used for hub microbial identification. Additionally, LEfSe analysis was utilized to identify differentially microbial taxa and functional pathways among three groups. 【Result】A total of 2625245 Clean reads were obtained from cecal content sequencing of 35 Guizhou local chickens, resulting in 727 operational taxonomic units(OTUs) clustered at a threshold of 97% sequence similarity. At the phylum level, dominant taxa in the cecal microbiota of three Guizhou local chicken breeds included Bacteroidetes, Firmicutes, Proteobacteria, Synergistetes and Spirochaetes. The top five genera in terms of relative abundance were Bacteroides, Rikenellaceae_RC9_gut_group, Phascolarctobacterium, Faecalibacterium and Desulfovibrio. Each of the Wumeng black-bone chicken, Qiandongnan Xiaoxiang chicken and Yaoshan chicken yielded 160, 144, and 149 OTUs for interaction network analysis, respectively. The stability index of cecal microbial interaction networks were ranked as Yaoshan chicken(44.22%)>Qiandongnan Xiaoxiang chicken(41.76%)>Wumeng black-bone chicken(37.56%), while the complexity of networks were ranked as Qiandongnan Xiaoxiang chicken>Wumeng blackbone chicken>Yaoshan chicken. Oscillospira was the highest scoring hub microbiota in the cecal microbial interaction networks of Qiandongnan Xiaoxiang chicken and Yaoshan chicken, while Lactobacillus was a hub microbiota in Qiandongnan Xiaoxiang chicken, and Rikenellaceae_RC9_gut_group was hub microbiota in Wumeng black-bone chicken. LEfSe analysis revealed that Wumeng black-bone chicken had 15 characteristic OTUs, and their predicted functional pathways involved in amino acid biosynthesis, 2-oxocarboxylic acid metabolism, arginine biosynthesis; Qiandongnan Xiaoxiang chicken enriched 2 characteristic OTUs, their predicted functional pathways involved in photosynthesis and streptomycin biosynthesis; Yaoshan chicken had 6 characteristic OTUs associated with lipopolysaccharide biosynthesis functional pathway. 【Conclusion】Breed is an important factor influencing the composition and structure of cecal microbiota of Guizhou chicken breeds. Specific microbes are enriched in the ceca of different chicken breeds. For instance, Rikenellaceae_RC9_gut_group, Ruminiclostridium and Odoribacter are enriched in the ceca of Wumeng black-bone chicken; Megamonas, Rikenellaceae_RC9_gut_group, Phascolarctobacterium are enriched in Yaoshan chicken; and Desulfovibrio and Faecalibacterium are enriched in Qiandongnan Xiaoxiang chicken.