The adaptation of bacterial lineages to local environmental conditions creates the potential for broader genotypic diversity within a species, which can enable a species to dominate across ecological gradients because of niche flexibility. The genus Polynucleobacter maintains both free-living and symbiotic ecotypes and maintains an apparently ubiquitous distribution in freshwater ecosystems. Subspecies-level resolution supplemented with metagenome-derived genotype analysis revealed that differential functional constraints, not geographic distance, produce and maintain strain-level genetic conservation in Polynucleobacter populations across three geographically proximal riverine environments. Genes associated with cofactor biosynthesis and one-carbon metabolism showed habitat specificity, and protein-coding genes of unknown function and membrane transport proteins were under positive selection across each habitat. Characterized by different median ratios of nonsynonymous to synonymous evolutionary changes (dN/dS ratios) and a limited but statistically significant negative correlation between the dN/dS ratio and codon usage bias between habitats, the free-living and core genotypes were observed to be evolving under strong purifying selection pressure. Highlighting the potential role of genetic adaptation to the local environment, the two-component system protein-coding genes were highly stable (dN/dS ratio, < 0.03). These results suggest that despite the impact of the habitat on genetic diversity, and hence niche partition, strong environmental selection pressure maintains a conserved core genome for Polynucleobacter populations. IMPORTANCE Understanding the biological factors influencing habitat-wide genetic endemism is important for explaining observed biogeographic patterns. Polynucleobacter is a genus of bacteria that seems to have found a way to colonize myriad freshwater ecosystems and by doing so has become one of the most abundant bacteria in these environments. We sequenced metagenomes from locations across the Chicago River system and assembled Polynucleobacter genomes from different sites and compared how the nucleotide composition, gene codon usage, and the ratio of synonymous (codes for the same amino acid) to nonsynonymous (codes for a different amino acid) mutations varied across these population genomes at each site. The environmental pressures at each site drove purifying selection for functional traits that maintained a streamlined core genome across the Chicago River Polynucleobacter population while allowing for site-specific genomic adaptation. These adaptations enable Polynucleobacter to become dominant across different riverine environmental gradients.