Background: The role of fatty acids (FAs) on mammographic density (MD) is unclear, and available studies are based on self-reported dietary intake. Objectives: This study assessed the association between specific serum phospholipid fatty acids (PLFAs) and MD in premenopausal women. Methods: The cross-sectional study DDM-Madrid recruited 1392 Spanish premenopausal women, aged 39-50 y, who attended a screening in a breast radiodiagnosis unit of Madrid City Council. Women completed lifestyle questionnaires and FFQs. Percentage MD was estimated using a validated computer tool (DM-Scan), and serum PLFA percentages were measured by GC-MS. Multivariable linear regression models were used to quantify the association of FA tertiles with MD. Models were adjusted for age, education, BMI, waist circumference, parity, oral contraceptive use, previous breast biopsies, and energy intake, and they were corrected for multiple testing. Results: Women in the third tertile of SFAs showed significantly higher MD compared with those in the first tertile (beta(T3vsT1) = 7.53; 95% CI: 5.44, 9.61). Elevated relative concentrations of palmitoleic (beta(T3vsT1) = 3.12; 95% CI: 0.99, 5.25) and gondoic (beta(T3vsT1) = 2.67; 95% CI: 0.57, 4.77) MUFAs, as well as high relative concentrations of palmitelaidic (beta(T3vsT1) = 5.22; 95% CI: 3.15, 7.29) and elaidic (beta(T3vsT1) = 2.69; 95% CI: 0.59, 4.79) trans FAs, were also associated with higher MD. On the contrary, women with elevated relative concentrations of n-6 (omega-6) linoleic (beta(T3vsT1) = -5.49; 95% CI; -7.62, -3.35) and arachidonic (beta(T3vsT1) = -4.68; 95% CI: -6.79, -2.58) PUFAs showed lower MD. Regarding desaturation indices, an elevated palmitoleic to palmitic ratio and a low ratio of oleic to steric and arachidonic to dihomo. gamma-linolenic acids were associated with higher MD. Conclusions: Spanish premenopausal women with high relative concentrations of most SFAs and some MUFAs and trans FAs showed an increased MD, whereas those with high relative concentrations of some n-6 PUFAs presented lower density. These results, which should be confirmed in further studies, underscore the importance of analyzing serum FAs individually.