Bovine mycoplasma mastitis is highly transmittable and hard to treat by chemotherapy. It causes severe economic loss and is considered a major problem for milk production. Mycoplasma canadense is one of the causative agents of mycoplasma mastitis. A primer set to detect M. canadense was developed based on single nucleotide polymorphism-specific loop-mediated isothermal amplification. Using this primer set, 10 fg M. canadense DNA corresponding to the DNA amount of ~13 cells was detected within 40 min. Cross-reactivity with other bovine Mycoplasma spp., Acholeplasma laidlawii, and mastitis-related bacteria was not observed when ≤1 pg DNA was applied. These results would provide a basis for validating future experiments with spiked-milk and field samples for the development of rapid detection of M. canadense.