The plasmids in gut microbiomes have the potential to contribute to the microbiome community, as well as human health and physiology. Nevertheless, this niche remains poorly explored. In general, most microbiome studies focus on urban-industrialized groups, but here, we studied semi-isolated groups from South America and Africa, which would represent a link between ancestral and modern human groups. Based on open metagenomic data, we characterized the set of plasmids, including their genes and functions, from the gut microbiome of the Hadza, Matses, Tunapuco, and Yanomami, semi-isolated groups with a hunter, gather or subsistence lifestyle. Unique plasmid clusters and gene functions for each human group were identified. Moreover, a dozen plasmid clusters circulating in other niches worldwide are shared by these distinct groups. In addition, novel and unique plasmids harboring resistance (encompassing six antibiotic classes and multiple metals) and virulence (as type VI secretion systems) genes were identified. Functional analysis revealed pathways commonly associated with urban-industrialized groups, such as lipopolysaccharide biosynthesis that was characterized in the Hadza gut plasmids. These results demonstrate the richness of plasmids in semiisolated human groups’ gut microbiome, which represents an important source of information with biotechnological/pharmaceutical potential, but also on the spread of resistance/virulence genes to semi-isolated groups.