Oligosaccharides represent one of the main components of human milk, and they have been assigned important biological functions for newborns. Qualitatively and quantitatively, their presence in milk is strictly related to the expression of the mother's Se and/or Le genes, on the basis of which 4 different milk groups have been described. The aim of the study was to provide new data on the oligosaccharide composition of preterm milk in relation to the 4 groups. High-pH anion-exchange chromatography was used to quantify levels of 23 oligosaccharides and lactose in 252 milk samples collected from 63 mothers during the first month of lactation and to identify the 4 milk groups. Substantial differences in oligosaccharide contents were found within the groups and were strictly related to the presence or absence of specific fucosyl-oligosaccharides. The highest concentration was found in group 1 (>20 g/L), the lowest level was found in group 4 (∼10 g/L), and intermediate values were observed in groups 2 and 3. No statistically significant differences in lactose concentrations were observed among the groups. Our data confirm lower lactose concentrations in preterm milk, compared with term milk, and they provide the first detailed characterization of oligosaccharides in preterm milk, demonstrating important differences in oligosaccharide contents in the 4 groups. These differences might exert an influence on several biological functions that are particularly important for preterm infants and currently are attributed to milk oligosaccharides.