In this study, we conducted a survey of the cytochrome b (cytb) gene of Babesia gibsoni (B. gibsoni) isolated from clinical cases to determine the prevalence of potential atovaquone (ATV)-resistant variants. Ninety-two blood samples were collected from naturally B. gibsoni infected dogs. The cytb nucleotide sequence was determined by direct sequencing. Twelve non-synonymous amino acid substitutions were identified in cytb. The principal ATV-resistant substitution, M121I, was detected in three cases. This survey determined that potentially ATV-resistant B. gibsoni strains are present in dogs in Japan.