The microbiota of the mammalian gut is a complex ecosystem, the composition of which is greatly influenced by host genetics and environmental factors. The goal of this comparative study was to investigate the influence of area of habitation, species, age and sex on intestinal microbiota composition of the three lemur species Eulemur fulvus, E. rubriventer, and E. rufifrons.MethodsFecal samples were collected from wild lemurs across Madagascar, and microbial composition was determined using next generation sequencing of PCR-amplified 16S ribosomal RNA gene fragments. Results Fecal microbiota of all three lemur species was dominated by members of the phyla Firmicutes, Bacteroidetes, Proteobacteria and Cyanobacteria. Consistent with reports from other primate species, the most predominant phyla were Firmicutes (43±6.4% [s.d.]) and Bacteroidetes (30.3±5.3%). The microbial composition was strongly associated with geographical area of habitation, with up to 19.9% of the total variation in microbial composition being explained by this factor in the E. fulvus population distributed across three sampling locations. In turn, differences observed in fecal microbiota of sympatric lemur species were less pronounced, as was the impact of the factors sex and age. Conclusions Our findings showed that among the studied factors the geographical region of habitation had the strongest influence on intestinal microbiota of congeneric lemur species. This suggests adaptation of microbiota to natural differences in forest composition, climate variations, and corresponding differences in the available diet in different geographical locations of Madagascar.