The effect of linseed oil (LO) supplementation to red clover silage (RCS)- or corn silage (CS)-based diets on rumen microbiota composition was studied in 12 rumen-cannulated lactating dairy cows in a 4 × 4 Latin square design (35-d periods) with a 2 × 2 factorial arrangement of treatments. Rumen liquid and solid samples were collected on d 18 of each period. DNA was extracted and V4 region of 16S rRNA was amplified and subjected to illumina paired-end sequencing. In both rumen liquid and solids, the LO supplementation reduced bacterial α-diversity in CS-based diet but not in RCS (P < 0.05). The LO supplementation altered the β-diversity of rumen microbiota in both CS- and RCS-based diets though the magnitude of shift was greater in CS. The LO supplementation reduced the abundances of several bacterial phyla including Actinobacteria, Chloroflexi, Fibrobacteres, Firmicutes, Plantomycetes, SR1, Spirochetes and Tenericutes, and increased the abundances of Bacterioidetes, Elusimicrobia, Lentisphaerae, Proteobacteria and Synergistetes in the CS-based diet (P < 0.05) but did not change the proportion of the abovementioned phyla in the RCS-based diet. The LO supplementation reduced methanogenic Methanobrevibacters in both diets, but the magnitude of depression found to be greater in CS compared with RCS. The LO supplementation increased Methanosphaera population with a greater magnitude in the CS-based diet compared with RCS (P < 0.05). The LO supplementation also negatively affected the proportion of several beneficial rumen bacteria including Bifidobacteria, Fibrobacter and Mollicutes with greater magnitude in the CS-based diet compared with RCS (P < 0.05). Data mechanistically explains how LO supplementation differentially affected CH₄ emission and animal production in CS- vs. RCS-based diets observed in the parallel study to this. The LO supplementation more effectively reduced the CH₄ emission (26%) in CS vs. RCS (9%), which might be due to greater reduction in Methanobrevibacters. Also, LO supplementation reduced DMI, fiber digestion, and yields of milk fat and protein only in the CS-based diet, which might be due to its negative effect on fibrolytic populations.