Objective The aim of this study was to examine differences in fatty acid concentrations in colostrum of women with and without gestational diabetes mellitus (GDM). The effect of GDM on fatty acid composition of colostrum is not fully understood, although rates of GDM are increasing globally. Methods A prospective case–control study was conducted of postpartum women with and without GDM. Gas chromatographic analysis was conducted to examine differences in colostral fatty acids of the colostrum samples of 29 women with and 34 without GDM. Results Analyses of the fatty acid composition revealed significantly higher concentrations of four essential ω-6 polyunsaturated fatty acids—γ-linolenic, eicosatrienoic, arachidonic, and docosatetraenoic—in the colostrum of GDM women compared with non-GDM women. Timing of collection influenced saturated medium chain fatty acid and monounsaturated fatty acid levels. Conclusions Differences in concentrations of ω-6 fatty acids but not in dietary linoleic fatty acid or ω-3 fatty acids suggest that altered concentrations are attributed to changes in specific endogenous metabolic pathways. Implications of higher concentrations of ω-6 fatty acids in the colostrum of women with GDM have yet to be determined. Timing of colostrum collection is critical in determining colostral fatty acid and metabolite concentrations.