Dietary intakes of linoleic acid (LA) have increased, including in women of reproductive age. Changes in maternal gut microbiome have been implicated in the metabolic adaptions that occur during pregnancy. We aimed to investigate if consumption of a diet with elevated LA altered fecal microbiome diversity prior to and during pregnancy. Female Wistar Kyoto rats consumed a high LA diet (HLA: 6.21% of energy) or a low LA diet (LLA: 1.44% of energy) for 10 weeks prior to mating and during pregnancy. DNA was isolated from fecal samples prior to pregnancy (embryonic day 0 (E0)), or during pregnancy at E10 and E20. The microbiome composition was assessed with 16S rRNA sequencing. At E0, the beta diversity of LLA and HLA groups differed with HLA rats having significantly lower abundance of the genera Akkermansia, Peptococcus, Sutterella and Xo2d06 but higher abundance of Butyricimonas and Coprococcus. Over gestation, in LLA but not HLA rats, there was a reduction in alpha diversity and an increase in beta diversity. In the LLA group, the abundance of Akkermansia, Blautia, rc4.4 and Streptococcus decreased over gestation, whereas Coprococcus increased. In the HLA group, only the abundance of Butyricimonas decreased. At E20, there were no differences in alpha and beta diversity, and the abundance of Roseburia was significantly increased in the HLA group. In conclusion, consumption of a HLA diet alters gut microbiota composition, as does pregnancy in rats consuming a LLA diet. In pregnancy, consumption of a HLA diet does not alter gut microbiota composition.