<h3>Background and aim</h3> Polyunsaturated fatty acids (PUFAs) are essential for early human neurodevelopment. Since breast milk represents the main dietary source of PUFAs for preterm infants, its fatty acid composition is of particular interest. Breast milk fatty acid composition fluctuates from one woman to another, by duration of gestation and stage of lactation, and among countries. As PUFA composition of breast milk from Western Canadian mothers of preterm infants has been previously described, the aim of this pilot study was to describe the compositional variations of PUFAs in breast milk of Eastern Canadian mothers of preterm infants over the first 6 weeks of lactation. <h3>Methods</h3> Samples of breast milk were collected on the 7th, 21st, and 42nd day of lactation from 40 Eastern Canadian mothers who had given birth to preterm infants of gestational age less than 30 weeks. Lipids were extracted using the Folch method (Folch <i><i>et al</i></i>., 1957). Thereafter, the method of direct transesterification was performed (Lepage and Roy, 1986) and PUFAs were quantified by gas chromatography. <h3>Results</h3> Throughout the study, fatty acids from the n-3 and n-6 families, linoleic acid (C18:2n-6), α-linolenic acid (C18:3n-3), and eicosapentaenoic acid (C20:5n-3) remained stable, whereas arachidonic acid (C20:4n-6) and docosahexaenoic acid (C22:6n-3) decreased significantly. <h3>Conclusion</h3> Results on the compositional variations of PUFAs in breast milk over the first weeks of lactation are somewhat similar to results of previous European studies. However, values of PUFAs in preterm breast milk of Eastern Canadian mothers are lower than those of Western Canadian mothers.