Mutations in Babesia gibsoni mitochondrial cytochrome b genes—specifically at the M128 position—are associated with resistance to atovaquone, which can lead to treatment failure. This study sought to determine M128 mutation prevalence in B gibsoni in blood from dogs in North America. The study also evaluated how many of these patients had wild-type cytochrome b in initial blood samples and M128 mutations in follow-up samples. Prevalence of the M128 mutation in the 173 dogs tested was 3.5%; incidence of new cytochrome b mutations in the 43 dogs with follow-up testing was 12.1%. American Staffordshire/American pit bull terriers comprised 74% of dogs infected with B gibsoni in this study. The authors concluded that the cytochrome b mutation is not common enough to warrant pretreatment mutation screening prior to therapy.