Agalinis auriculata (Orobanchaceae) is a state-listed species in Illinois and is rare throughout its range in the midwestern United States. In recent years, research has been conducted on the biology of this hemiparasitic plant in order to understand better what factors should be included in the development of sound preservation practices. Our objective in this study was to assess genetic variation using allozyme and RAPD markers for Illinois populations of A. auriculata and two of its more common congeners, A. purpurea and A. tenuifolia. A total of 13 allozyme loci and 112 RAPD markers were scored for the studied species. Overall, lower percent polymorphic loci/markers were observed in A. auriculata than in its congeners. Analyses of molecular variance on RAPD markers showed that most of the genetic variation was within populations. In addition, the allozyme and RAPD UPGMA and RAPD principle coordinates analyses separated the three species of Agalinis. The genetic results of this study in conjunction with ecological studies of A. auriculata in Illinois serve as a springboard for the conservation of the species, not only in Illinois but also throughout its range.