Lyme disease is the most commonly reported vector-borne disease in the United States. constitute an additional zoonotic pathogen whose public health impact and diversity continue to emerge. Rapid, sensitive, and specific detection of these and other vector-borne pathogens remains challenging, especially for patients with persistent infections. This report describes an approach for DNA extraction and PCR testing for the detection of spp. and spp. from dry blood spot (DBS) specimens from human patients. The present study included extraction of DNA and PCR testing of DBS samples from 105 patients with poorly defined, chronic symptoms labeled as Lyme-Like Syndromic Illness (LLSI). spp. DNA was detected in 20/105 (19%) and spp. DNA was detected in 41/105 (39%) patients with LLSI. Neither group of organisms was detected in DBS samples from 42 healthy control subjects. spp. 16S-23S rRNA internal transcribed spacer sequences were highly similar to ones previously identified in yellow flies, lone star ticks, a human patient from Florida, mosquitoes in Europe, or and strains from honeybees. These human strains may represent new genetic strains or groups of human pathogenic species of . The 41 spp. gene sequences obtained from human patients suggested the presence of four different species, including , , and /-like strains. These results suggest that specific aspects of the DBS DNA extraction and PCR approach enabled the detection of spp. and spp. DNA from very small amounts of human whole blood from some patients, including specimens stored on filter paper for 17 years.