A multiplex primer-dependent nucleic acid amplification and detection method for diagnosing at least two tick-borne diseases from the group consisting of Lyme, babesiosis and anaplasmosis in a sample from a human or other mammal, comprising the steps of: a. contacting DNA from said sample with primer-dependent amplification reagents that include at least two of the following: (i) a primer pair, having the respective sequences of SEQ ID Nos:12 and 13, for a recA gene sequence of Borrelia that differs among B. burgdorferi, B. afzelii, and B. garinii, and one or a complement of Borrelia -specific molecular beacon probes having the SEQ ID Nos: 5 or 30 and capable of distinguishing among those species; (ii) a primer pair, having the respective sequences of SEQ ID Nos: 14 and 15 or of SEQ ID Nos: 24 and 25, for a conserved BmTPK gene sequence of B. microti, and a Babesia -specific molecular beacon probe having the sequence of SEQ ID No: 16, 28, or 6; and (iii) a primer pair, having the respective sequences of SEQ ID Nos: 18 and 19, or of SEQ ID Nos: 26 and 27, for a conserved APH 1387 gene sequence of A. phagocytophilum, and an Anaplasmosis -specific molecular beacon probe having the sequence of SEQ ID No: 20, 29, or 9, wherein said primer pairs define amplicons that are 70-300 base pairs in length, and wherein each different molecular beacon probe is labeled with a spectrally distinguishable fluorescent or luminescent signaling moiety, b. performing said primer-dependent amplification to obtain amplified products of said gene sequences, and c. detecting said amplified products, thereby identifying said subject as infected or not infected with said at least two pathogens. a. contacting DNA from said sample with primer-dependent amplification reagents that include at least two of the following: (i) a primer pair, having the respective sequences of SEQ ID Nos:12 and 13, for a recA gene sequence of Borrelia that differs among B. burgdorferi, B. afzelii, and B. garinii, and one or a complement of Borrelia -specific molecular beacon probes having the SEQ ID Nos: 5 or 30 and capable of distinguishing among those species; (ii) a primer pair, having the respective sequences of SEQ ID Nos: 14 and 15 or of SEQ ID Nos: 24 and 25, for a conserved BmTPK gene sequence of B. microti, and a Babesia -specific molecular beacon probe having the sequence of SEQ ID No: 16, 28, or 6; and (iii) a primer pair, having the respective sequences of SEQ ID Nos: 18 and 19, or of SEQ ID Nos: 26 and 27, for a conserved APH 1387 gene sequence of A. phagocytophilum, and an Anaplasmosis -specific molecular beacon probe having the sequence of SEQ ID No: 20, 29, or 9, wherein said primer pairs define amplicons that are 70-300 base pairs in length, and wherein each different molecular beacon probe is labeled with a spectrally distinguishable fluorescent or luminescent signaling moiety, (i) a primer pair, having the respective sequences of SEQ ID Nos:12 and 13, for a recA gene sequence of Borrelia that differs among B. burgdorferi, B. afzelii, and B. garinii, and one or a complement of Borrelia -specific molecular beacon probes having the SEQ ID Nos: 5 or 30 and capable of distinguishing among those species; (ii) a primer pair, having the respective sequences of SEQ ID Nos: 14 and 15 or of SEQ ID Nos: 24 and 25, for a conserved BmTPK gene sequence of B. microti, and a Babesia -specific molecular beacon probe having the sequence of SEQ ID No: 16, 28, or 6; and (iii) a primer pair, having the respective sequences of SEQ ID Nos: 18 and 19, or of SEQ ID Nos: 26 and 27, for a conserved APH 1387 gene sequence of A. phagocytophilum, and an Anaplasmosis -specific molecular beacon probe having the sequence of SEQ ID No: 20, 29, or 9, b. performing said primer-dependent amplification to obtain amplified products of said gene sequences, and c. detecting said amplified products, thereby identifying said subject as infected or not infected with said at least two pathogens. <EOS>