Although gut microbiome contributes to the traits and diseases of its host as much as its own genes, current researches based on fecal microbiome have resulted many inconsistent and often contradictory data on how gut microbiome affects its host. In this study using α- and β-diversity analyses on mouse gastrointestinal (GI) contents, we showed that the compositions of GI microbiome were location specific and, more importantly, fecal microbiome was very different from any microbiome of the GI tract, contrary to the baseline assumption in the large majority of contemporary microbiome research work. Our discovery on the microbiome discrepancy between feces and the GI tract seems to explain the unanswered ambiguity in current microbiome researches based on fecal analyses, suggesting the importance of reliable tool for the analysis of microbiome and host traits. Funding Statement: This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (Grant No. HI18C2039). Declaration of Interests: Authors declare no competing interests. Ethics Approval Statement: All animal care and use protocols were performed strictly in accordance with the ethical guidelines of the Ethics Committee of the Chonbuk National University Laboratory Animal Center in accordance with the ‘Guide for the Care and Use of Laboratory Animals’, published by the National Research Council and endorsed by the ARRIVE Guidelines.