Rickettsia parkeri is an Amblyomma-associated, spotted fever group Rickettsia species that causes an eschar-associated, febrile illness in multiple countries throughout the Western Hemisphere. Many other rickettsial species of known or uncertain pathogenicity have been detected in Amblyomma spp. ticks in the Americas, including Rickettsia amblyommii, "Candidatus Rickettsia andeanae" and Rickettsia rickettsii. In this study, we utilized an immunoproteomic approach to compare antigenic profiles of low-passage isolates of R. parkeri and R. amblyommii with serum specimens from patients with PCR- and culture-confirmed infections with R. parkeri. Five immunoreactive proteins of R. amblyommii and nine immunoreactive proteins of R. parkeri were identified by matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry. Four of these, including the outer membrane protein (Omp) A, OmpB, translation initiation factor IF-2, and cell division protein FtsZ, were antigens common to both rickettsiae. Serum specimens from patients with R. parkeri rickettsiosis reacted specifically with cysteinyl-tRNA synthetase, DNA-directed RNA polymerase subunit alpha, putative sigma (54) modulation protein, chaperonin GroEL, and elongation factor Tu of R. parkeri which have been reported as virulence factors in other bacterial species. Unique antigens identified in this study may be useful for further development of the better serological assays for diagnosing infection caused by R. parkeri.