The objective was to examine the effect of the dose and viability of Saccharomyces cerevisiae (SC) on the ruminal microbial population of lactating dairy cows. Four ruminally cannulated lactating cows (284 ± 18 DIM) were used in a study with a 4 × 4 Latin square design with four 21-d periods. Cows were fed a TMR supplemented with no SC (Control) or a low dose of live SC (5.7 × 10⁷ cfu/day; LLY), a high dose of live SC (6.0 × 10⁸ cfu/day), or a high dose of killed SC (6.0 × 10⁸ cfu/day before heating at 80°C). Ruminal fluid collected 0, 2, 4, 6, 8 and 10 h after the morning feeding on d 21 was strained through cheesecloth to obtain liquid (LF) and solid (SF) fractions. Microbial diversity was examined by high throughput Illumina MiSeq sequencing of the V4 region of the 16S rRNA gene. Populations of select ruminal bacteria and protozoa were also quantified by qPCR. Data were analyzed using the GLIMMIX procedure of SAS. In the SF and LF, Prevotella was the most abundant genus (23.8 and 49.1%) followed by Fibrobacter (10.7 and 1.5%) and Succinivibrionaceae (7.1 and 4.6%), respectively. Supplemental LLY increased the prevalence of Butyrivibrio (P = 0.11) and Ruminococcus (P = 0.04) in the LF. Supplementing with live instead of dead SC increased the prevalence of Ruminococcus in the LF and SF (P ≤ 0.05). Supplemental killed SC decreased the prevalence of Lachnospiraceae (P = 0.06) and Coprococcus (P = 0.13) in the SF and increased those of Ruminobacter and Porphyromonadaeae in the SF and LF (P ≤ 0.12, respectively). The qPCR results showed that in the SF, supplementing with LLY or with live instead of killed SC increased the prevalence of F. succinogenes (P = 0.14 and 0.04, respectively). Supplemental killed SC decreased the prevalence of protozoa (P = 0.05) and B. fibrosolvens (P = 0.14). In the LF, supplemental LLY decreased the prevalence of R. albus (P = 0.12) and increased that of S. ruminantium (P = 0.14). Unlike the high dose of live or killed SC, feeding the low SC dose or live instead of killed SC increased the prevalence of fiber-degrading bacteria.