Nutritional factors are known to influence the development of the gastrointestinal (GI) tract in preruminants. However, the effect of different lipid sources on the bacterial communities in the GI tract of young calves has not been well described. Therefore, the aim of this study was to utilize high-throughput sequencing to investigate the GI microbiota of preruminant calves fed supplemental polyunsaturated fatty acids. Calves (n = 8) were fed milk (4 L per d) plus 3% supplemental oil containing either palm oil or a combination of conjugated linoleic acid and flaxseed oil in a ratio of 1:2 (CLA/FLAX) for 50 d beginning 3–6 d after birth. After euthanasia, digesta samples from the rumen, omasum, abomasum, duodenum, cecum, and large intestine (LI) were collected. DNA was extracted using the Qiagen DNA Stool Mini kit and bacterial DNA amplified using primers targeting the V1-V3 hypervariable region of the 16S rRNA gene. Amplicons were sequenced using an Illumina MiSeq v3 paired-end 300-bp protocol for 600 cycles. Sequences were processed and classified using the custom python application dbcAmplicons. Principal coordinate analysis at the genus level revealed 3 clusters: (1) rumen, omasum, abomasum; (2) duodenum; (3) cecum and LI. All 3 clusters had large proportions of reads that could not be classified at the genus level: 1) 39.9% ± 1.9; 2) 76.2% ± 5.4; 3) 56.4% ± 2.6. Clusters 1 and 2 were enriched in Prevotella (24.4% ± 1.6 and 7.2% ± 2.7, respectively) and Succinivibrio (8.7% ± 1.4 and 2.8% ± 2.1, respectively) while Cluster 3 had similar proportions of Prevotella (6.2% ± 2.2), Bacteroides (6.2% ± 1.9), and Oscillibacter (4.3% ± 0.6). Using generalized linear mixed models, rumen, omasum, and abomasum bacterial communities from calves fed palm oil had greater (P < 0.05) abundances of Succinivibrio (13.10% ± 1.9 vs 4.1% ± 1.0), Oscillibacter (2.7% ± 0.4 vs 1.1% ± 0.3), and Paraprevotella (2.5% ± 0.4 vs 1.4% ± 0.3), and lower (P < 0.05) abundances of Prevotella (20.4% ± 1.8 vs 28.4% ± 2.1) as compared with those from calves fed CLA/FLAX. The implications of different ruminal bacterial composition on growth and tissue development in preruminant calves warrant further research.