Human granulocytic anaplasmosis (HGA) is a tick borne infection caused by Anaplasma phagocytophilum. HGA cases in South Korea have been identified since the first report in 2014. In this study, we investigated the serological response in 594 clinical samples of patients with acute febrile illness and molecular characteristics of A. phagocytophilum clinical isolates obtained from HGA patients. In serological test for A. phagocytophilum, 7.91% (47/594 cases) were positive for IgG and Ig M and 13 of 47 cases showed seroconversion. In the detection rate of the 16S rRNA, msp2(p44), and ankA, genes were showed 3.68% (14/380 cases) for A. phagocytophilum- specific 16S rRNA gene. Phylogenetic analysis of three clinical isolates demonstrated high sequence similarity (98.58– 100%) with A. phagocytophilum 16S rRNA sequences identified from public databases. Analysis of the msp2(p44) gene showed highly variable similarity rates (7.24–98.85%) even within isolated countries and host ranges. These results provide clues into the bacterial characterization of A. phagocytophilum originating from Korean patients, providing useful guidance for treatment and improving clinical outcomes.