Significance The diversification of host-associated microbial communities depends on barriers to gene flow, imposed by confinement to different hosts or by niche partitioning within single hosts. However, most gut microbiomes are too complex to disentangle the diversification processes. Taking advantage of the simple gut microbiomes of social bees, we demonstrate that bee gut bacteria have diversified both between host species and within single host species through the acquisition of different ecological niches within the same gut. Our study further shows that gut microbiomes differ in spatial distributions within the same host, possibility due to adaptation to specified nutritional niches, such as urea utilization.