Background <p><i>Rickettsia parkeri</i> sensu stricto, a causative agent of spotted fever rickettsiosis, is spread via the bite of infected ticks within the Amblyomma maculatum complex group. In the United States of America (USA), Am. maculatum Koch, 1844 is the primary vector for R. parkeri; however, Amblyomma americanum (Linnaeus, 1758) and Dermacentor variabilis (Say, 1821) have demonstrated potential to transmit R. parkeri under laboratory conditions. In this study, we investigate the genetic differences between R. parkeri detected in Am. maculatum, the primary enzootic vector, and potential secondary vectors – Am. americanum and De. variabilis.</p> Methods <p>Using Nanopore long-read amplicon sequencing, we compared four R. parkeri genes amplified from naturally infected <i>Am. maculatum, Am. americanum</i>, and De. variabilis collected in the USA. Three R. parkeri genes associated with potential virulence factors were sequenced: outer membrane protein A (OmpA/sca0), outer membrane protein B (OmpB/sca5), and surface cell antigen 4 (<i>gene D/sca4</i>). One species-level gene target was used for species confirmation: 16S ribosomal RNA gene (16S).</p> Results <p>Differences in cellular-entry and pathogen chromosomal genes (OmpA, OmpB, and 16S) were detected within the different tick species. No differences were noted in the cell-to-cell mediated transfer gene (<i>gene D</i>) between tick species.</p> Conclusions <p>This preliminary study suggests that infection in Am. americanum may lead to changes in R. parkeri genes responsible for pathogen-host cell attachment and replication processes, but once established in a host cell, R. parkeri transfer between cells is unlikely to be impacted.</p>