ABSTRACT Background DHA (22:6n–3) is essential for neurodevelopment in children, and its concentration in human breast milk is historically high in Japan. Dietary patterns in Japan might affect the fatty acid (FA) composition among lactating mothers. Objectives This study aimed to characterize the composition of milk FAs and to identify any dietary and sociodemographic factors associated with the variability of DHA concentration in breast milk in the Japanese population. Methods This cross-sectional study was performed as part of the Japanese Human Milk Study. Milk FAs were analyzed by GC at 1–6 mo postpartum, and maternal diet was estimated using an FFQ, including 11 types and cooking methods of seafoods, and the use of DHA supplements. The association of milk DHA with maternal diet and sociodemographic factors was investigated. Results Milk FA concentrations were measured in 78 mothers, including 24 who routinely used DHA supplements. The DHA concentration in milk (overall median: 0.62%; IQR: 0.47%–0.78%) was higher in women who took DHA supplements than in women who had never used DHA supplements (0.74%compared with 0.55%; P = 0.011). A linear regression model showed the association of milk DHA concentration with maternal dietary intake of grilled fish (β ± SE: 0.006 ± 0.003; standardized β: 0.234; r2 = 0.232, P = 0.036) after adjustment for DHA supplementation status, maternal and infant age, maternal BMI, and infant birth weight. Other FA concentrations were consistent, whereas caproic acid (6:0), undecylic acid (11:0), pentadecylic acid (15:0), palmitoleic acid (16:1n–7), and vaccenic acid (18:1n–7) varied by DHA supplementation status. Conclusions The DHA concentration in human milk may be influenced by maternal grilled fish consumption and frequent DHA supplementation in lactating Japanese women. Milk DHA concentrations may reflect a dietary habit in Japanese mothers. This trial was registered at www.umin.ac.jp/ctr as UMIN000015494.