Glucose-5-phosphate dehydrogenase (G6PD) deficiency is an X-linked genetic disorder that affects red blood cells’ metabolism. This retrospective study aimed to investigate the prevalence and characteristics of G6PD testing in Korea. Data were collected from laboratory information systems between July 2021 and June 2022. A total of 5193 patients (1722 males and 3471 females) with a median age of 55.1 years (interquartile range, IQR 44.6 to 64.5) were tested for whole blood G6PD, with 1.6% of tests performed on patients of non-Korean ethnicity. The majority of tests were performed in hospitals (37.7%) or local clinics (34.5%). Interestingly, no female children were tested for whole blood G6PD during the study period. The prevalence of decreased G6PD activity (<7.9 U/g Hb) was 0.4% (19/5111 Koreans and 2/82 non-Koreans), and only seven male patients with G6PD deficiency (<30% of the male median) were identified, with ages ranging from 4.8 months to 50.2 years. No female patients with G6PD deficiency were found. Further research is necessary to determine the clinical significance of G6PD test results and monitor their use.