Dermacentor variabilis is the predominant tick species in Nebraska and is presumed to be the primary vector of Rickettsia rickettsii associated with cases of Rocky Mountain spotted fever (RMSF). Interestingly, RMSF cases in Nebraska have increased on a year-to-year basis, yet the prevalence of R. rickettsii in D. variabilis ticks has not been established for Nebraska. Here we sought to set a baseline for the prevalence of R. rickettsii and other spotted fever group (SFG) rickettsiae harbored by D. variabilis ticks. Over a 3-yr period, D. variabilis were collected along the Platte River in south central Nebraska. Individual tick DNA was analyzed using endpoint PCR to identify ticks carrying SFG rickettsiae. In total, 927 D. variabilis were analyzed by PCR and 38 (4.1%) ticks tested positive for SFG rickettsiae. Presumptive positives were sequenced to identify the Rickettsia species, of which 29 (76%) were R. montanensis, 5 (13%) were R. amblyommatis, 4 (11%) were R. bellii, and R. rickettsii was not detected. These data indicate that R. rickettsii is likely at a low prevalence in south central Nebraska and spillover of R. amblyommatis into D. variabilis is likely occurring due to the invasive lone star tick (Amblyomma americanum). In addition, our data suggest that R. montanensis and R. amblyommatis could be associated with the increase in SFG rickettsiae infections in Nebraska.This information will be of value to clinicians and the general public for evaluating diagnosis of disease- and risk-associated environmental exposure, respectively.