23Background: Human gut microbiome profiles have been associated with 24 human health and disease. These profiles have been defined based on 25 microbes' taxonomy and more recently, on their functionality. Human groups 26 that still maintain traditional modes of subsistence (hunter-gatherers and rural 27 agriculturalists) represent the groups non-impacted by urban-industrialized 28 lifestyles, and therefore study them provide the basis for understanding the 29 human microbiome evolution. The Yanomami is the largest semi-nomadic 30 hunter-gatherer group of the Americas, exploring different niches of the Amazon 31 rainforest in Brazil and Venezuela. In order to extend the analysis of this unique 32 and diverse group, we focused on the gut microbiome of the Yanomami from 33Brazil and compared with those from Venezuela, and also with other traditional 34 groups from the Amazon, considering taxonomic and functional profiles. 35 Results: A diversity of taxonomic biomarkers were identified to each South 36 American traditional group studied, including the two Yanomami groups, despite 37 their overall similarity in the taxonomic gut microbiome profiles. Broader levels 38 of functional categories poorly discriminated traditional and urban-industrialized 39 groups. Interestingly, a diversity was observed with the stratification of these 40 categories, clearly segregating those groups. The Yanomami/Brazil gut 41 microbiome presented unique functional features, such as a higher abundance 42 of gene families involved in regulation/cell signaling, motility/chemotaxis, and 43 virulence, contrasting with the microbiomes from the Yanomami/Venezuela and 44 other groups. 45 Conclusions: Our study revealed biomarkers, taxonomic and functional 46 differences between the gut microbiome of Yanomami/Brazil and 47 Yanomami/Venezuela individuals. This intra-Yanomami group diversity was 48 accessed due to the increase number of individuals and group studied. These 49 differences may reflect their semi-nomadic behavior, as well as, the local and 50 seasonal diversity of the vast rainforest niche they explore, despite their shared 51 cultural and genetic background. Overall, their microbiome profiles are shared 52 with South American and African traditional groups, probably due to their 53 lifestyle. The unique features identified within the Yanomami highlight the bias 54 imposed by underrepresented sampling, and factors such as variations over 55 space and time (seasonality) that impact, mainly, the hunter-gatherers. 56 Therefore, to reach knowledge about human microbiome variations and their 57 implications in human health, it is essential to enlarge data concerning the 58 number of individuals, as well as the groups representing different lifestyles. 59 60