We evaluated the serological and molecular prevalence of selected organisms in 145 dogs during late spring (May/June) of 2005 and in 88 dogs during winter (February) of 2007 from the Hopi Indian reservation. Additionally, in 2005, 442 ticks attached to dogs were collected and identified as Rhipicephalus sanguineus. Infection with or exposure to at least one organism was detected in 69% and 66% of the dogs in May/June 2005 and February 2007, respectively. Exposure to spotted fever group (SFG) rickettsiae was detected in 66.4% (2005) and 53.4% (2007) of dogs, but rickettsial DNA was not detected using polymerase chain reaction. Active Ehrlichia canis infection (by polymerase chain reaction) was identified in 36.6% (2005) and 36.3% (2007) of the dogs. E. canis infection was associated with SFG rickettsiae seroreactivity (p < 0.001). Anaplasma platys DNA was detected in 8.3% (2005) and 4.5% (2007) of the dogs. Babesia canis and Bartonella vinsonii berkhoffii seroprevalences were 6.7% and 1% in 2005, whereas in 2007 prevalences were 0% and 1.1%, respectively. No Bartonella spp., Ehrlichia chaffeensis, or Ehrlichia ewingii DNA was detected. Dogs on this Hopi Indian reservation were most frequently infected with E. canis or A. platys; however, more than half of the dogs were exposed to a SFG-Rickettsia species.