Description: Tibetan Chickens should have unique gastrointestinal microbiota because of their particular habitats. Thus, the aim of this study was to investigate the cecal microbiota of Tibetan Chickens from five typical high-altitude regions of China. Lohmann egg-laying hens (LMs) and Daheng broiler chickens (DHs) were chosen as controls. The cecal bacterial populations of Tibetan Chickens were surveyed by high-throughput sequencing (HTS) of the bacterial 16S rRNA hypervariable region V3-V4 (16S rRNAV3-V4) combined with community-fingerprinting analysis of the 16S rRNA gene based on polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The results revealed that the majority of cecal microbiota differed between the Tibetan Chicken and LM/DH. The microbial communities in the cecum were composed of 16 phyla, 28 classes, 36 orders, 57 families, 101 genera, and 189 species. Represented phyla were Bacteroidetes (〉47%), Firmicutes (〉18.8%), Spirochaetae (〉0.3%), and Proteobacteria (〉0.4%). Bacteroides and the RC9 gut group were the two most abundant genera. There were relatively more Christensenellaceae , Subdoligranulum , Spirochaeta , and Treponema in Tibetan Chickens, whereas there were more Phascolarctobacterium , Faecalibacterium , Megamonas , and Desulfovibrio in LMs and DHs. The cecal microbiota of Tibetan Chicken have slightly diverged due to exposure to different geographic environments. Differences in the intestinal bacterial communities of Tibetan Chicken and LM/DH were noted. The cecal bacterial populations of Tibetan Chickens were surveyed by high-throughput sequencing (HTS) of the bacterial 16S rRNA hypervariable region V3-V4 (16S rRNAV3-V4) combined with community-fingerprinting analysis of the 16S rRNA gene based on polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The cecal microbiota of Tibetan Chicken have slightly diverged due to exposure to different geographic environments. Differences in the intestinal bacterial communities of Tibetan Chicken and LM/DH were noted.