| Summary: | Even though the epidemiology of tick-borne agents (TBA) in dogs has been extensively investigated around the world, the occurrence, vectors involved, and molecular identity of these agents in cats remains elusive in many regions. Among TBA, <i>Ehrlichia</i>, <i>Anaplasma</i>, <i>Babesia</i>, <i>Cytauxzoon</i>, and <i>Hepatozoon</i> are responsible for diseases with non-specific clinical signs in cats, making essential the use of molecular techniques for accurate diagnosis and proper treatment. The present work aimed to investigate the occurrence and molecular identity of tick-borne agents (<i>Ehrlichia</i>, <i>Anaplasma</i>, <i>Babesia</i>/<i>Theileria</i>, <i>Cytauxzoon</i>, and <i>Hepatozoon</i>) in cats from southeastern (states of São Paulo (SP) and Minas Gerais (MG)) and northern (state of Rondônia (RO)) Brazil. For this purpose, 390 blood samples were collected from domiciled cats in MG (<i>n</i> = 155), SP (<i>n</i> = 151), and RO(<i>n</i> = 84) states, submitted to DNA extraction and PCR assays for <i>Ehrlichia</i> spp. (<i>dsb</i> gene), <i>Anaplasma</i> spp. (<i>rrs</i> gene), piroplasmids (18S rRNA gene), and <i>Hepatozoon</i> spp. (18S rRNA gene), sequencing, and phylogenetic inferences. The overall positivity for <i>Anaplasma</i> spp., <i>Ehrlichia</i> spp., <i>Babesia/Theileria</i> spp., <i>Cytauxzoon</i> spp., and <i>Hepatozoon</i> spp. were 7.4% (12.3% (MG) and 6.6% (SP)), 2% (4.5% (MG) and 0.6% (SP)), 0.7% (0.6% (MG), 0.6% (SP) and 1.2% (RO)), 27.2% (41.9% (MG), 24.5% (SP) and 4.8% (RO), and 0%, respectively. The phylogenetic analysis grouped the obtained sequences with ‘<i>Candidatus</i> Anaplasma amazonensis’, <i>A. platys</i>, <i>B. vogeli</i>, and <i>Cytauxzoon</i> sp. previously detected in wild felids from Brazil. qPCR specific for <i>E. canis</i> based on the <i>dsb</i> gene confirmed the molecular identity of the detected ehrlichial agent. The present study expanded the list and geographical distribution of hemoparasites in cats. ‘<i>Candidatus</i> Anaplasma amazonensis’, recently detected in sloths from northern Brazil, was described for the first time in cats. This is the first report of piroplasmids infecting cats in northern Brazil. Coinfection by <i>Cytauxzoon</i> and other TBA (<i>Ehrlichia, Anaplasma</i>, and <i>B. vogeli</i>) reported in the present study raises the need for veterinary practitioners’ awareness of cats parasitized by multiple TBA.
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