PCR amplification targeting the 16S rRNA gene was used to test individuals with and without extensive arthropod and animal contact for the possibility of hemotropic mycoplasma infection. The prevalence of hemotropic mycoplasma infection (4.7%) was significantly greater in previously reported cohorts of veterinarians, veterinary technicians, spouses of veterinary professionals, and others with extensive arthropod exposure and/or frequent animal contact than in a previously reported cohort of patients examined by a rheumatologist because of chronic joint pain or evidence of small-vessel disease (0.7%). Based upon DNA sequence analysis, a Mycoplasma ovis-like species was the most prevalent organism detected; however, infection with "Candidatus Mycoplasma haematoparvum" and a potentially novel, but incompletely characterized, hemotropic Mycoplasma species was also documented. Historical exposure to animals and arthropod vectors that can harbor hemotropic Mycoplasma spp. should be considered during epidemiological investigations and in the evaluation of individual patients.