Maternal diet has decisive influence on the fatty acid composition of human milk. Fifteen years ago, we found outstandingly low contribution of docosahexaenoic acid (DHA) to human milk in a small group of Hungarian mothers. The major aim of the present study was to investigate whether DHA status in human milk in Hungary changed during the last 15 years. We aimed to examine the fatty acid composition of human milk at three different stages of lactation (3rd day, 6th week, and 6th month) in healthy Hungarian mothers. Fatty acid composition of human milk lipids was determined by gas chromatograph with flame ionization detector. Contribution of arachidonic acid to the fatty acid composition of human milk significantly decreased during lactation (0.91 [0.38] in colostrum, 0.53 [0.17] at 6th week, and 0.46 [0.13] at 6th month, p < 0.01). The contribution of DHA significantly decreased from colostrum to the 6th week of lactation (0.29 [0.12] and 0.14 [0.04], p < 0.01), without further changes by 6 months (0.12 [0.10]).The contribution of DHA to the fatty acid composition of mature human milk in Hungarian mothers is still among the lowest values ever reported in the literature.