Human granulocytic ehrlichiosis (HGE) is an emerging tick-borne disease recently recognized in the United States. The blacklegged tick, Ixodes scapularis Say, is the principle vector in the eastern United States. The disease has been commonly reported in the northeastern and upper midwestern states; however, suitable vectors and reservoir hosts exist in the southeast. To assay the prevalence of the HGE agent in vector ticks, we screened 818 individual I. scapularis from 15 locations in South Carolina, Georgia, and Florida using nested polymerase chain reaction, which targets the HGE agent 16S rRNA gene. Prevalence among locations ranged from 0 to 20%. The overall average prevalence of 15 sites was 1.6% (n = 818). Verification by sequencing the 16S rDNA from the positive samples showed 99.8–100% nucleotide identities with the sequences of the HGE agent in GenBank. These results were supported by the phylogenetic analysis using 16S rDNA sequences.