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. Fecal samples were collected on d 24–29 of each period and pooled. DNA was extracted and V4 region of 16S rRNA was amplified and subjected to Illumina paired-end sequencing. DNA was extracted and V4 region of 16S rRNA was amplified and subjected to illumina paired-end sequencing. The feces from cows fed RCS-based diets had greater microbial α-diversity compared with CS, but LO supplementation did not affect α-diversity of microbial community. In contrast, the LO supplementation altered the β-diversity of fecal microbota only in the CS-based diet (P = 0.001). Among 17 bacteria phyla found in fecal samples, the LO supplementation reduced both Actinobacteria and Bacteroidetes populations and increased Fimicutes under the CS-based diet (P < 0.05). However, under the RCS-based diet, the LO supplementation showed no significant effect on any of bacterial phyla. In CS-based fed cows, LO supplementation suppressed abundances of 2 genera from Bacteriodales family, unclassified RF16 and unclassified Rikenellaceae, as well as the genus SHD231 in Anaerolinaceae family (P < 0.05). The 16S rRNA sequencing generated taxa belonged to Euryarchaeota in which no archaeal genera were found to be associated with LO supplementation. Data suggest that LO supplementation only affected fecal microbiota in the CS-based diet and that the source of the basal forage diet influences the effectiveness of LO supplementation and composition of microbiota even in the hindgut of dairy cows.