A highly conserved 357-bp segment of the 16S ribosomal RNA gene (16S rDNA) of Borrelia burgdorferi sensu lato and the correspondent 358-bp segment of the Borrelia miyamotoi gene were amplified by a single pair of nested polymerase chain reaction (PCR) primers for detection, and the amplicons were used as the templates for direct Sanger DNA sequencing. Reliable molecular diagnosis of these borreliae was confirmed by sequence alignment analysis of the hypervariable regions of the PCR amplicon, using the Basic Local Alignment Search Tool (BLAST) provided by the GenBank. This methodology can detect and confirm B. burgdorferi and B. miyamotoi in blood samples of patients with off-season spirochetemia of low bacterial density. We found four B. miyamotoi infections among 14 patients with spirochetemia, including one patient co-infected by both B. miyamotoi and B. burgdorferi in a winter month when human exposure to tick bites is very limited in the Northeast of the U.S.A. We conclude that sensitive and reliable tests for these two Borrelia species should be implemented in the microbiology laboratory of hospitals located in the disease-endemic areas, for timely diagnosis and appropriate treatment of the patients at an early stage of the infection to prevent potential tissue damages.