We report Babesia microti genomic sequences with multiple mutations in the atovaquone-target region of cytochrome b, including a newly identified Y272S mutation, plus one mutation of undetermined significance in the azithromycin-associated ribosomal protein L4. The parasite was sequenced from an immunocompromised patient on prophylactic atovaquone for Pneumocystis pneumonia prior to diagnosis of babesiosis.