The objective of this study was to compare fermentation profile, and microbial diversity from rumen samples collected using rumen cannula or stomach tube in lactating dairy cows. Three ruminally cannulated lactating dairy cows were used as rumen fluid donors. Treatments were rumen fluid collected from Stomach tube (ST) or from rumen cannula (C). The pH of rumen contents was measured immediately after collection and rumen samples were analyzed for volatile fatty acid (VFA), ammonia-N (NH3-N) concentration, and metagenome. Data were analyzed using GLIMMIX procedure of SAS. Rumen pH was greater for ST compared to C (6.87 vs 6.25). However, NH3-N (15.2 vs 10.6 mg/dL) and total VFA (121.8 vs 95.5 mM) was greater for C compared to ST. The bacterial community was dominated by the phylum Bacteroidetes, Firmicutes, and Cyanobacteria, regardless of the rumen fluid collection method. However, the abundance of Bacteroidetes was greater while abundance of Firmicutes was lower for ST compared with C. The rumen fluid collection methods had no effects on Chao 1 and Shannon index. In conclusion, molar proportion of individual VFA, Chao1, and Shannon index were similar while rumen pH, NH3-N, and total VFA were affected by the rumen fluid collection method.