Gaylussacia brachycera (box huckleberry) is a slow-growing, dwarf evergreen member of the family Ericaceae that is native to eight states in the eastern United States. It is a rare plant with conservation status in several states of critically imperiled (S1). Botanists have been intrigued by this enigmatic native plant since it was discovered in 1796 in Virginia. One of the mysteries of this species is whether plants in a colony arose from different genotypes or are clonal. The species reproduces primarily by means of underground runners and appears to be self-sterile, so sexual reproduction within isolated colonies could be limited. Using molecular markers, we tested samples taken from three of the best-known colonies in Pennsylvania and one in Tennessee. Based on 104 polymorphic markers, we found that one of the Pennsylvania colonies contained two genotypes among 11 samples tested; one Pennsylvania colony contained three genotypes among five samples tested; and the other two colonies exhibited no variation among the 8–10 samples tested. This study represents the first time that molecular markers have been used in a systematic assay to determine the existence of variation among individuals within a colony of box huckleberry.