Comparison of culture, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), and 16S rRNA genomic sequencing to identify mastitis pathogens was the objective. All quarter milks submitted to The Dairy Authority (TDA) laboratory on one day were streaked (10 µL) onto Columbia blood agar and MacConkey agar. Plates with colonies were number coded, paraffin sealed and shipped overnight with cold packs to the University of Missouri (MU). Culture at TDA identified bacteria to genus level except for speciation of S. aureus and E. coli. PCR was negative for Mycoplasma spp. At MU, a MALDI-TOF mass spectrometer tested colonies in duplicate. Comparison with the Biotyper database of known bacteria produced scoring between 1.7 and 1.99 for genus level identification and ≥2.0 for species level. 16S rRNA colony lysate PCR products were Sanger sequenced, and sequences were compared with GenBank data using nucleotide-BLAST at MU. All microbiologists were blind to other results. Culture and MALDI-TOF tested 181 isolates; 16S rRNA tested 179 (2 lost during storage). In accordance with culture, S. aureus and E. coli agreement was to species level, all others to genus level. This was a test of agreement, not sensitivity or specificity (no “gold standard”). Overall agreement between the 3 diagnostic methods was 87% (155/179). Agreement between MALDI-TOF and 16S rRNA was 98% (176/179). That assumes agreement for 29 isolates called E. coli by culture and MALDI-TOF that 16S rRNA defined as E. coli or Shigella spp. Most bacteria were identified with good agreement among all 3 methods by McNemar’s test, including 94% (80/85) of isolates defined by culture as Staph spp., with 22 isolates defined by culture as S. aureus, Enterobacter spp., Klebsiella spp., Pasteurella spp., or T. pyogenes showing 100% agreement among all 3 methods. Many members of the dairy industry are comfortable using either bacterial culture or MALDI-TOF for routine milk bacteria diagnosis; 16S rRNA is mainly a research tool. The results suggest that all 3 methods are valuable tools for the dairy industry.