Tongue diagnosis is a unique method in traditional Chinese medicine (TCM). This is the first investigation on the association between traditional tongue diagnosis and the tongue coating microbiome using next-generation sequencing. The study included 19 gastritis patients with a typical white-greasy or yellow-dense tongue coating corresponding to TCM Cold or Hot Syndrome respectively, as well as eight healthy volunteers. An Illumina paired-end, double-barcode 16S rRNA sequencing protocol was designed to profile the tongue-coating microbiome, from which approximately 3.7 million V6 tags for each sample were obtained. We identified 123 and 258 species-level OTUs that were enriched in patients with Cold/Hot Syndromes, respectively, representing “Cold Microbiota” and “Hot Microbiota”. We further constructed the tongue microbiota-imbalanced networks associated with Cold/Hot Syndromes. The results reveal an important connection between the tongue-coating microbiome and traditional tongue diagnosis, and illustrate the potential of the tongue-coating microbiome as a novel holistic biomarker for characterizing patient subtypes.