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Background  Composition and maintenance of the microbiome is vital to gut homeostasis. However, there is limited knowledge regarding the impact of high doses of radiation, which can occur as a result of cancer radiation therapy, nuclear accidents or intentional release of a nuclear or radioactive weapon, on the composition of the gut microbiome. Therefore, we sought to analyze alterations to the gut microbiome of nonhuman primates (NHPs) exposed to high doses of radiation. Fecal samples were collected from 19 NHPs (Chinese rhesus macaques,  Macaca mulatta ) 1 day prior and 1 and 4 days after exposure to 7.4 Gy cobalt-60 gamma-radiation (LD 70–80/60 ). The 16S V4 rRNA sequences were extracted from each sample, followed by bioinformatics analysis using the QIIME platform.    Results  Alpha Diversity (Shannon Diversity Index), revealed no major difference between pre- and post-irradiation, whereas Beta diversity analysis showed significant differences in the microbiome after irradiation (day + 4) compared to baseline (pre-irradiation). The Firmicutes/Bacteriodetes ratio, a factor known to be associated with disruption of metabolic homeostasis, decreased from 1.2 to less than 1 post-radiation exposure.  Actinobacillus, Bacteroides, Prevotella (Paraprevotellaceae family) and Veillonella  genera were significantly increased by more than 2-fold and  Acinetobacter  and  Aerococcus  genus were decreased by more than 10-fold post-irradiation. Fifty-two percent (10/19) of animals exposed to radiation demonstrated diarrhea at day 4 post-irradiation. Comparison of microbiome composition of feces from animals with and without diarrhea at day 4 post-irradiation revealed an increase in  Lactobacillus reuteri  associated with diarrhea and a decrease of  Lentisphaerae  and  Verrucomicrobioa  phyla and  Bacteroides  in animals exhibiting diarrhea. Animals with diarrhea at day 4 post-irradiation, had significantly lower levels of Lentisphaere and Verrucomicrobia phyla and Bacteroides genus at baseline before irradiation, suggesting a potential association between the prevalence of microbiomes and differential susceptibility to radiation-induced diarrhea.    Conclusions  Our findings demonstrate that substantial alterations in the microbiome composition of NHPs occur following radiation injury and provide insight into early changes with high-dose, whole-body radiation exposure. Future studies will help identify microbiome biomarkers of radiation exposure and develop effective therapeutic intervention to mitigate the radiation injury.    Supplementary Information  The online version contains supplementary material available at 10.1186/s12866-021-02146-w.

Changes in the gut microbiome community of nonhuman primates following radiation injury