Microorganisms in surface water constructed wetland (CW) play crucial roles in pollutant removal. How-ever, little is known about the diversity and structure of microbial community in surface water CW. Theinfluential factors regulating microbial community diversity and structure remain poorly known. In thepresent study, Illumina high-throughput sequencing was used to characterize bacterial and archaealcommunities in three lab-scale vertical-flow CW (VF-CW) systems with different substrate materials(fine gravel, steel slag or natural zeolite). The depth-related change of archaeal and bacterial communityrichness, diversity and structure occurred in these three VF-CW systems. Microbial diversity in threeVF-CW systems showed the similar depth-related change pattern, but microbial richness illustrated thedifferent change pattern. Substrate type had a profound effect on bacterial richness but only a slighteffect on bacterial diversity. Archaeal richness and diversity were affected by substrate type and wetlanddepth. Moreover, archaeal community had much lower richness and diversity than bacterial commu-nity. Archaeal and bacterial community structure was regulated by substrate type and wetland depth.Proteobacteria and Acidobacteria were the most abundant bacterial phyla, while Euryarchaeota was thepredominant archaeal phylum.