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Background: Tracking the spread of antibiotic resistant bacteria is critical to reduce global morbidity and mortality associated with human and animal infections. There is a need to understand the role that wild animals in maintenance and transfer of antibiotic resistance genes (ARGs)., Methods: This study used metagenomics to identify and compare the abundance of bacterial species and ARGs detected in the gut microbiomes from sympatric humans and wild mouse lemurs in a forest-dominated, roadless region of Madagascar near Ranomafana National Park. We examined the contribution of human geographic location toward differences in ARG abundance and compared the genomic similarity of ARGs between host source microbiomes., Results: Alpha and beta diversity of species and ARGs between host sources were distinct but maintained a similar number of detectable ARG alleles. Humans were differentially more abundant for four distinct tetracycline resistance-associated genes compared to lemurs. There was no significant difference in human ARG diversity from different locations. Human and lemur microbiomes shared 14 distinct ARGs with highly conserved in nucleotide identity. Synteny of ARG-associated assemblies revealed a distinct multidrug-resistant gene cassette carrying dfrA1 and aadA1 present in human and lemur microbiomes without evidence of geographic overlap, suggesting that these resistance genes could be widespread in this ecosystem. Further investigation into intermediary processes that maintain drug-resistant bacteria in wildlife settings is needed., Competing Interests: Timothy D. Read and Patricia C. Wright are Academic Editors for PeerJ., (©2024 Talbot et al.)