Mycoplasma bovisis a major cause of bovine mastitis. Intermittent shedding of the organism for many months is a feature of cows with intramammary infection. A dairy farm in Japan experienced a mastitis outbreak caused by M. bovisin 2016, as well as 2 additional outbreaks and 1 case in 2020–2021. The causative strains in the 3 outbreaks shared a common and identical genetic feature, the insertion of a transposase gene at the same site within the phosphate acetyltransferase-2 gene. Additionally, all isolates were genotyped to closely related sequence types (ST21 and ST141) by multilocus sequence typing, and had similar pulsopatterns by pulsed-field gel electrophoresis. Our results indicate that infection with the same causative strain remained in this herd and environment for 4 y. Treatment with fluoroquinolones, guided by antimicrobial susceptibility test results, eliminated M. bovisfrom 16 of 20 M. bovis–infected cows, as confirmed by culture and somatic cell counts. However, mastitis caused by other bacteria occurred in 9 M. bovis–free cows within 2 mo of the last treatment.