The environmental microbiome study was designed to decipher microbial diversity of the International Space Station surfaces in terms of spatial and temporal distributions using 16S and ITS iTag Illumina sequencing. We hypothesized that the microbial population of environmental surfaces changes in time due to astronauts xe2 x80 x99 activity and might be location specific. The environmental samples were collected with the polyester wipes from eight different locations in the ISS during two consecutive sampling sessions (three months apart). The specific objective was to unveil the viable microbial diversity of each location during two separate sessions in terms of abundance and richness of the communities. The International Space Station (ISS) as a closed built environment has its own environmental microbiome which is shaped by microgravity radiation and limited human presence. The microbial diversity associated with ISS environmental surfaces was investigated during this study. Polyester wipes and contact slides were used for sampling of eight various surface locations on the ISS at different time periods. The samples were retrieved and analyzed immediately upon the return to the Earth (via Soyuz TMA-14M or Dragon capsule from SpaceX). After surface sample collection contact slides containing nutrient media for the growth of bacteria and fungi were incubated at 25 xcb x9aC. The polyester wipes were processed to measure microbial burden (R2A Blood Agar and Potato Dextrose Agar) and recover cultivable bacteria as well as fungi. Subsequently viable microbial burden was assessed using Adenosine Triphosphate (ATP) assay and quantitative polymerase chain reaction (PCR) methods after propidium monoazide (PMA) treatment. The 16S-tag and metagenome analyses were used to elucidate viable microbial diversity. The cultivable bacterial population yield from the polyester wipes was very high (5 to 7-logs) when compared with the contact slides (102 to 103 CFU/m2). The PMA-qPCR analysis showed considerable variation of viable bacterial population (105 to 109 16S rDNA gene copies/m2) among locations sampled. Unlike contact slides polyester wipes cover much larger sample surface (~1 m2) and produce much more reliable results of the microbial diversity of the ISS covering both cultivable and non-cultivable species. The cultivable total and viable microbial diversity was determined utilizing state-of-the art molecular techniques. The implementation of the PMA assay before DNA extraction allowed distinguishing viable microorganisms which is crucial for determining their role to the crew health the ISS maintenance and the general knowledge of the closed environmentally controlled built systems.