Lotmaria passim Schwarz (Trypanosomatidae) is a recently described trypanosome parasite of honey bees in the continental United States, Europe, and Japan. We developed a multiplex PCR technique using PCR primers specific for L. passim or Crithidia species to distinguish L. passim from C. mellificae Langridge and McGhee. We report the presence of L. passim in Hawaii and American Samoa for the first time. More importantly, this multiplex PCR will be a useful diagnostic technique for screening honey bee samples for the presence of this pathogenic trypanosome.