This study aimed to identify bacterial isolates from the Tigris River in Samarra using molecular techniques. The results revealed bacterial species from the genera Escherichia, Enterobacter, Aeromonas, Klebsiella, and Pseudomonas. Identification was confirmed via 16S rRNA sequence analysis, showing high similarity (97–100%) with reference strains in the NCBI database. Point and insertion mutations were particularly noted in Klebsiella pneumoniae and Pseudomonas aeruginosa, suggesting environmental stress. The study highlights the value of molecular tools in environmental monitoring and emphasizes the need for continuous water quality assessment due to the potential presence of multidrug-resistant bacteria suggesting the influence of environmental stress and pollution on the genetic structure of these bacteria.