The gut microbiota is intricately associated with the onset and progression of colorectal cancer (CRC), leading to significant interest in developing prevention and treatment strategies that leverage gut microbiota. In this study, we collected 57 samples from 19 CRC patients, comprising cancerous tissue, paracancerous tissue, and normal mucosa. Utilizing metagenomic sequencing and bioinformatics analysis, we identified differences in the microbiomes and their functional characteristics across the various tissue types. The results indicated that species such as were predominantly found in normal tissues, while was enriched in paracancerous tissue, and was prevalent in cancerous tissues. Furthermore, the microbial functions exhibited variability among the different tissue types. Random forest analysis suggested that may be implicated in the onset and progression of colorectal cancer. We also classified the patients into three subgroups based on the anatomical location of the colorectum: right-sided colon, left-sided colon, and rectum. The subgroup analysis revealed that the microbiota enriched in normal mucosa and paracancerous tissue varied across different anatomical sites. These findings not only elucidate the characteristics of the microbiomes in the normal mucosa, paracancerous tissue, and cancerous tissues of CRC patients, thereby providing new potential targets for clinical diagnosis and treatment, but also contribute to the existing microbiome data pertinent to CRC research.IMPORTANCEThis study provides crucial insights into the relationship between gut microbiota and colorectal cancer (CRC) by analyzing microbial communities in different tissue types and anatomical locations of CRC patients. We identified distinct microbial signatures, such as in normal tissues and in cancerous tissues, indicating location-specific microbiomes with unique functional attributes. These findings suggest potential new biomarkers or therapeutic targets for CRC. The observed microbiota variations among right-sided colon, left-sided colon, and rectum cancers underscore the heterogeneity of CRC, pointing toward more personalized treatment strategies. By enhancing our understanding of the microbiome's role in CRC, this research paves the way for innovative diagnostic tools and targeted therapies tailored to individual patient profiles. This work is essential for advancing clinical approaches to CRC management.