Species are the fundamental unit of ecology and evolutionary biology. However, there is no universal definition of a species that spans all domains of life. Eukaryotic species are traditionally defined through reproductive isolation, where reproductive barriers result in adaptive traits being shared only within the isolated population. Therefore, discontinuities in gene flow in eukaryotes generally result in the formation of an ecologically and genotypically cohesive unit. In contrast, delineating asexual bacterial species is often hampered by extensive horizontal gene flow and is further confounded by the difficulty of identifying a bacteria’s ecological niche. Therefore, the working definition of a prokaryotic species is based on arbitrary standards of relatedness and often lacks a cohesive ecological niche. To address this problem, the ecological population theory, which defines bacterial ‘species’ as ecologically distinct populations delineated through discontinuities in total gene flow, was proposed. It postulates that, rather than a hindrance, the prevalence of horizontal gene flow, primarily through homologous recombination, plays a significant role in bacterial speciation. Horizontal gene flow allows gene-specific sweeps in bacterial populations, where adaptive genes are shared preferentially between recombining bacteria independent of their genomic background. Thus, discontinuities in horizontal gene flow can be utilised to demarcate a genotypically and ecologically cohesive bacterial species. Previous studies have demonstrated that ecological populations exist in heterotrophic bacteria. However, examining the generalisability of the ecological population theory requires understanding the role of horizontal gene flow in delineating populations from extreme bacterial communities, especially bacteria with limited metabolic redundancy due to their higher impact of deleterious consequences (e.g., pathway dysregulation by recombination of a maladaptive gene) during horizontal gene flow. Therefore, in this work, I utilised the chemolithoautotrophic hydrogen-oxidising Aquificota bacteria from natural populations as a model system to test the generalisability of the ecological population theory. Utilising a novel high-throughput isolation method, I isolated co-habiting Aquificota bacteria with minimal enrichment from geothermal springs. Sequencing and comparative analysis of the Aquificota genomes reveal intra-species genomic variations driven by varying degrees of horizontal gene flow and a population structure composed of co-occurring clonal lineages and recombinogenic bacteria. To examine whether these Aquificota populations exhibit ecological and evolutionary patterns akin to eukaryotic species, I investigated the changes in their population structure across four additional time points (1 year). Analysis of recent horizontal gene flow and mutations in Aquificota populations show that clonal lineages remain clonal across time, with minimal changes in their genomic background diversity. In contrast, recombining populations experience variation in intra-population diversity and a shift in the dominant genomic background across time—implying an intra-population turnover. Lastly, I also investigated the role of both non-homologous and homologous recombination in maintaining ecological populations in Aquificota by analysing the presence and function of mobile genetic elements (MGEs) in its different populations. The lack of spatial and functional overlap between MGEs and horizontally swept regions confirms that homologous recombination drives horizontal gene flow in Aquificota populations. This work demonstrated that ecological populations in Aquificota can be delineated through discontinuities in horizontal gene flow driven primarily by homologous recombination. Thus, ecological population theory can be a generalisable model for speciation and evolution in bacteria.