BACKGROUND: An understudied component of the diet, branched-chain fatty acids (BCFAs) are distinctive saturated fatty acids that may have an important influence on health. Human-milk fatty acid composition is known to differ worldwide, but comparative data are lacking on BCFAs. OBJECTIVE: We tested the hypotheses that concentrations of BCFAs in human milk differ between populations and are associated with maternal diet. DESIGN: We surveyed the BCFA composition of samples collected as part of a standardized, prospective study of human-milk composition. Mothers were enrolled from 3 urban populations with differing diets: Cincinnati, Ohio; Shanghai, China; and Mexico City, Mexico. Enrollment was limited to healthy mothers of term singleton infants. We undertook a cross-sectional analysis of milk from all women with samples at postpartum week 4 (n = 359; ∼120 women/site). Fatty acids were extracted from milk by using a modified Bligh-Dyer technique and analyzed by gas chromatography. Statistical analysis was performed by ANOVA and Tobit regression. For Cincinnati mothers, 24-h diet recalls were analyzed in relation to the individual BCFA concentrations measured in milk samples. RESULTS: Total BCFAs in milk differed by site, with the highest concentration in Cincinnati followed by Mexico City and Shanghai (mean ± SE: 7.90 ± 0.41, 6.10 ± 0.36, and 4.27 ± 0.25 mg/100 mL, respectively; P < 0.001). Site differences persisted after delivery mode, maternal age, and body mass index were controlled for. The individual concentrations of iso-14:0, iso-16:0, iso-18:0, anteiso-15:0, and anteiso-17:0 also differed between sites. Milk concentrations of iso-14:0 and anteiso-15:0 were associated with maternal intake of dairy; iso-16:0 was associated with maternal intakes of dairy and beef. CONCLUSIONS: BCFA concentrations in milk at 4 wk postpartum differed between mothers from Cincinnati, Shanghai, and Mexico City. Variations in human-milk BCFAs are influenced by diet. The impact of BCFAs on infant health warrants investigation.