Hot spring microbial mats represent intricate biofilms that establish self-sustaining ecosystems, hosting diverse microbial communities which facilitate a range of biochemical processes and contribute to the structural and functional complexity of these systems. While community structuring across mat depth has received substantial attention, mechanisms shaping horizontal spatial composition and functional structure of these communities remain understudied. Here, we explored the contributions of species source, local environment and species interaction to microbial community assembly processes in six microbial mat regions following a flow direction with a temperature decreasing from 73.3°C to 52.8°C. Surprisingly, we found that despite divergent community structures and potential functions across different microbial mats, large proportions of the community members (45.50%-80.29%) in the recipient mat communities originated from the same source community at the upper limit of temperature for photosynthetic life. This finding indicated that the source species were dispersed with water and subsequently filtered and shaped by local environmental factors. Furthermore, critical species with specific functional attributes played a pivotal role in community assembly by influencing potential interactions with other microorganisms. Therefore, species dispersal via water flow, environmental variables, and local species interaction jointly governed microbial assembly, elucidating assembly processes in the horizontal dimension of hot spring microbial mats and providing insights into microbial community assembly within extreme biospheres.