The fatty acid composition of mature human milk obtained from 40 Spanish women was analyzed by capillary gas chromatography. The women were from two regions in Spain, Navarre and Catalonia. Milk samples were collected between 20 and 30 days postpartum. The fatty acid composition was expressed as weight percentage (% wt/wt of all fatty acids detected with a C8 to C22 chain length). Monounsaturated fatty acids represent 41.97%, mostly 18:1 n-9/n-7 (38.39%). The second major fraction was formed by saturated fatty acids, 41.09%. Polyunsaturated fatty acid fraction (15.23%), included seven long chain polyunsaturated fatty acids (LCPs; 2.21%). Among LCPs, 1.6% accounted for the n-6 series and 0.64% for the n-3 series. LCPn-6/LCPn-3 ratio was 2.51. Mothers reporting a high fish consumption showed higher (p < 0.05) 22:6 n-3 and 20:5 n-3 content. The use of olive oil as the preferential fat source showed higher 18:1 n-9/n-7 and lower 18:2 n-6 content (p < 0.0001), while the use of sunflower oil instead of olive oil significantly (p < 0.0001) increased 18:2 n-6 and decreased 18:1 n-9/n-7. Regional differences (p < 0.05) were detected only for the n-6 LCP and the total LCP content. The higher n-6 LCP and total LCP content was found in Navarre. This could have been due to different diet habits, like higher egg consumption.