Preterm infants show postnatal deficits of long-chain polyunsaturated fatty acids (LCPUFAs) which are essential for adequate growth and neurodevelopment. Human milk is a primary source of fatty acids (FAs) for the preterm infant, and therefore, knowledge about milk FA levels is required to design appropriate supplementation strategies. Here, we expanded on our previous study (Nilsson et al., 2018, Acta Paediatrica, 107, 1020–1027) determining FA composition in milk obtained from mothers of extremely low gestational age (<28 weeks) infants on three occasions during lactation. There was a clear difference in FA composition in milk collected at Day 7 and milk collected at postmenstrual weeks (PMW) 32 or PMW 40. Notably, the proportion of LCPUFAs was low and declined significantly during milk maturation. These results strengthen previous data that the content of FAs required by the preterm infant is not supplied in sufficient amounts when the mother’s own milk is the sole source of these essential nutrients.