The use of oro-esophageal probes to collect rumen samples for sequencing and characterization of microbiome became a widely used procedure in the last few years. While the methodology represents a proxy for the rumen microbiota, remain unclear how diversity, richness, and structure of microbiome is affected by the different methods of collection and by the different fractions of rumen sample that can be obtained by a rumen fistula. Our objectives were to compare richness, diversity, and structure of rumen microbiome in samples collected using an oro-esophageal probe (oral), and solid, liquid and the mixed solid-liquid fractions collected using a rumen fistula in Holstein dairy cows. Rumen-fistulated, mid-lactation, multiparous Holstein dairy cows (n = 9) fed the same diet had samples collected using the oro-esophageal tube and the rumen fistula. The rumen fistula samples included liquid, solid, and mixed liquid-solid fractions. Genomic DNA was extracted, and the V3-V4 hypervariable region of the 16S rRNA gene was amplified and sequenced using the Illumina MiSeq platform. Data were processed using QIIME and analyzed using JMP. The Chao1 richness index did not differ (P = 0.46), while the solid and mixed liquid-solid sample had a greater Shannon diversity index (P = 0.03) when compared with oral and liquid samples. The most common phyla across all samples were Bacteroidetes, Firmicutes, Proteobacteria, Spirochaetes, Euryarchaeota, Fibrobacteres, Actinobacteria, and Tenericutes. Mean relative abundance (MRA) of Fibrobacteres was greater (P = 0.02) in solid and mixed liquid-solid samples when compared with liquid and oral samples. Prevotella, Bacteroides, Succiniclasticum, Treponema, Ruminococcus, Butyrivibrio, Dysgomonas, Blautia, and Pedobacter were the most prevalent genera. No differences in the 30 most prevalent genera MRA were detected among sampling types nor were there differences among rumen source and methods and samples in our discriminant analysis. In conclusion, solid and mixed-solid liquid samples had greater microbiome diversity and MRA for the phylum Fibrobacteres than oral and liquid samples.