To obtain unrecorded freshwater bacterial species in Korea, water and sediment samples were collected from streams, lakes, and wetland of the Han River watershed in 2020. Approximately 800 bacterial strains were isolated on R2A agar after aerobic or anaerobic incubation, and identified using 16S rRNA gene sequences. A total of 28 strains, with ≥98.7% 16S rRNA gene sequence similarity with validly published bacterial species but not reported in Korea, were determined to be unrecorded bacterial species in Korea. The unrecorded bacterial strains were phylogenetically diverse and belonged to four phyla, eight classes, 13 orders, 19 families, and 25 genera. The unreported species were assigned to Acetobacter, Alsobacter, Mesorhizobium, Prosthecomicrobium, and Microvirga of the class Alphaproteobacteria; Vogesella, Formosimonas, Aquincola, Massilia, Acidovorax, and Brachymonas of the class Betaproteobacteria; Pseudoxanthomonas, Thermomonas, Lysobacter, Enterobacter, Kosakonia, and Acinetobacter of the class Gammaproteobacteria; Sulfuricurvum of the class Epsilonproteobacteria; Mycolicibacterium, Agromyces, Phycicoccus, and Microbacterium of the class Actinobacteria; Paenibacillus of the class Bacilli; Clostridium of the class Clostridia; and Flavobacterium of the class Flavobacteriia. The details of the unreported species, including Gram reaction, colony and cell morphology, biochemical characteristics, and phylogenetic position are also provided in the description of the strains.