A total of 90 Acinetobacter isolates from freshwater and seawater in Gangjin Bay of Korea was investigated for the distribution of genomic species, antimicrobial resistance patterns and clonal relatedness. By amplified ribosomal DNA restriction analysis, eighty-nine Acinetobacter isolates were classified into 11 Acinetobacter genomic species. A. johnsonii (n=23) was the most prevalent, followed by A. baumannii (n=13), A. calcoaceticus (n=13), Acinetobacter genomic species 11 (n=10), A. phenon 6/ct13TU (n=9), A. junii (n=5), A. venetianus (n=5), Acinetobacter genomic species 17 (n=4), 14BJ (n=3), A. phenon 10/1271 (n=2), Acinetobacter genomic species 3 (n=1), and ungrouped (n=1). The majority of Acinetobacter genomic species were isolated from the site A and B, and some known nosocomial pathogens in the clinical environment were observed among them. Of the 11 antimicrobial drugs tested, several A. johnsonii isolates exhibited high-frequency resistance to a wide variety of antimicrobial agents, including ampicillin-sulbactam, piperacillin, ceftazidime, cefotaxime, and sulfamethoxazole (p < 0.001). Some Acinetobacter genomic species were resistant to currently used antibiotics but all isolates were susceptible to imipenem, amikacin, and tetracycline. Based on the results of antimicrobial resistance pattern and phylogenetic analysis, 23 A. johnsonii isolates were classified into 19 pulsotypes. In conclusion, there was a significant difference in the distribution of Acinetobacter species between freshwater and seawater. Predominance of A. johnsonii strains was probably due to their ability to proliferate in the contaminated aquatic environment originated from local geographic features. Therefore, the waste effluent from animals and humans plays an important role in the distribution of Acinetobacter species in aquatic environment.