The molecular detection of Rickettsia parkeri in a Gulf Coast tick, Amblyomma maculatum, collected in Delaware represents the first evidence of the human pathogen R. parkeri associated with A. maculatum in the state. A total of five adult (2 male and 3 female) Gulf Coast ticks were collected from tick drags conducted during a two-day sampling event (21–22 May 2012) at Bombay Hook National Wildlife Refuge, near Smyrna, Delaware. All specimens were tested for the presence of Rickettsia with a genus-specific quantitative real-time polymerase chain reaction (qPCR) assay; one of the female specimens tested positive. This specimen was then assessed for the presence of Rickettsia parkeri and Candidatus Rickettsia andeanae by two species-specific qPCR assays. The presence of R. parkeri DNA was detected, whereas Candidatus R. andeanae DNA was not.