As part of the national recovery effort, endangered black-footed ferrets (Mustela nigripes) were reintroduced to the Cheyenne River Sioux Reservation in South Dakota, USA in 2000. Despite an encouraging start, numbers of ferrets at the site have declined. In an effort to determine possible causes of the population decline, we undertook a pathogen survey in 2012 to detect exposure to West Nile virus (WNV), canine distemper virus (CDV), plague (Yersinia pestis), tularemia (Francisella tularensis), and heartworm (Dirofilaria immitis) using coyotes (Canis latrans) as a sentinel animal. The highest seroprevalence was for WNV with 71% (20/28) of coyotes testing antibody-positive. Seroprevalence of CDV and plague were lower, 27% and 13%, respectively. No evidence of active infection with tularemia or heartworm was seen in the coyotes sampled. As this study did not sample black-footed ferrets themselves, the definitive cause for the decline of this population cannot be determined. However, the presence of coyotes seropositive for two diseases, plague and CDV, lethal to black-footed ferrets, indicated the potential for exposure and infection. The high seroprevalence of WNV in the coyotes indicated a wide exposure to the virus; therefore, exposure of black-footed ferrets to the virus is also likely. Due to the ability of WNV to cause fatal disease in other species, studies may be useful to elucidate the impact that WNV could have on the success of reintroduced black-footed ferrets as well as factors influencing the spread and incidence of the disease in a prairie ecosystem.