The main objectives of the current study were to estimate the bulk tank milk prevalence of 4 main mastitis pathogens (Staphylococcus aureus, Prototheca, Streptococcus agalactiae and Mycoplasma bovis) and determine the characteristics of herds that test positive. As part of the national study 374 farms were randomly selected from 10 provinces and visited between May and August in 2015 where an assessment of milking hygiene and other farm characteristics was undertaken. A sample of bulk tank milk was collected by the respective milk carriers between December 2015 and January 2016 and submitted for testing at CanWest DHI milk laboratory (Guelph, ON, Canada). All samples were tested using PathProof Mastitis Major 4 PCR Assay (Thermo Fisher Scientific Inc., Waltham, MA) according to manufacturer’s instructions. Production data for all Canadian farms were obtained from the provincial marketing boards. Based on the results of 372 milk samples, the apparent prevalence for each pathogen at the national level was as follows: 172 farms tested positive for S. aureus (46.2%; 95% CI: 41.4–51.6), 24 farms were positive for Prototheca (6.4%; 95% CI: 3.9–8.9), 2 farms were positive for M. bovis (0.5%; 95% CI: −0.2–1.3), and one farm was positive for S. agalactiae (0.3%; 95% CI: −0.3–0.8). Risk factor analysis for farms testing positive for S. aureus, based on multivariate logistical regression, determined that statistically significant characteristics were: pipeline milking systems (odds ratio 2.60; P < 0.0001), not tagging chronically infected animals (odds ratio 2.10; P = 0.010), and not fore-stripping (odds ratio 1.81; P = 0.039) while using dry cow therapy on every quarter of every cow was protective (odds ratio 0.45; P = 0.035). This is the first time Canada has been able to determine national prevalence data on these 4 common mastitis pathogens. The apparent prevalences are lower than those reported previously by individual regions or using non-random sampling.