Human milk (HM) provides infants with macro- and micronutrients needed for growth and development. Milk phospholipids are important sources of bioactive components, such as long-chain polyunsaturated fatty acids (LC-PUFA) and choline, crucial for neural and visual development. Milk from mothers who have delivered prematurely (<37 weeks) might not meet the nutritional requirements for optimal development and growth. Using liquid chromatography tandem-mass spectrometry, 31 phospholipid (PL) species were quantified for colostrum (2 weeks and ≤15 weeks) samples from mothers who had delivered preterm (n = 57) and term infants (n = 22), respectively. Both gestational age and age postpartum affected the PL composition of HM. Significantly higher concentrations (p < 0.05) of phosphatidylcholine (PC), sphingomyelin (SM) and total PL were found in preterm milk throughout lactation, as well as significantly higher concentrations (p < 0.002) of several phosphatidylethanolamine (PE), PC and SM species. Multivariate analysis revealed that PLs containing LC-PUFA contributed highly to the differences in the PL composition of preterm and term colostrum. Differences related to gestation decreased as the milk matured. Thus, gestational age may impact the PL content of colostrum, however this effect of gestation might subside in mature milk.