The aim of this study was to determine the typical incidence of micronuclei (MNi) in the peripheral blood lymphocytes of healthy middle-aged Koreans using the cytokinesis-block micronucleus cytome (CBMN-Cyt) assay. Non-smoking, low-risk alcohol-drinking healthy Korean men and women aged 30 to 59 years were recruited. Participants were divided into three groups according to age, i.e., 30 to 39, 40 to 49, and 50 to 59 years. Fifty participants were included in each age group, for a total of 300 participants. DNA damage was measured based on the number of binucleated (BN) cells with MNi, nucleoplasmic bridges (NPBs), and nuclear buds (NBUDs) using the CBMN-Cyt assay. The frequencies of BN cells with MNi in men were 14.0±4.9 (mean±SD) in 30-39year olds, 20.0±6.1 in 40-49year olds, and 21.7±7.6 in 50-59year olds. In women, they were 19.7±7.1 in 30-39year olds, 28.7±11.2 in 40-49year olds, and 31.9±12.9 in 50-59year olds. MNi and NPBs scores were higher in females than in males. The elder groups showed higher MNi frequencies for both genders, and the NPB frequency was higher in elder groups than younger groups, but only for males. Based on a regression analysis of the CBMN-Cyt parameters, MNi frequencies showed a positive relationship with age for both genders. BMI and blood vitamin B concentration were not significantly associated with CBMN-Cyt parameters, except vitamin B6 levels, which were positively associated with MNi scores in males. These results provide the standard frequencies of MNi, NPBs, and NBUDs in peripheral blood lymphocytes in middle-aged Korean individuals with healthy lifestyles. In this group, CBMN-Cyt assay parameters varied according to gender and age; however, BMI and micronutrient levels were not significantly associated with assay parameters.