Tipularia discolor is a terrestrial woodland orchid of the eastern United States that exhibits variable spatial patterns. The genetic diversity of several populations in Maryland, Delaware, and Virginia was examined using ISSR markers. Low levels of genetic variation were detected, yet four distinct genotypes were determined. A Mantel test revealed that genetic diversity did not correlate with spatial distance (Mantel t-test = −0.349; p = 0.364). An Analysis of Molecular Variance (AMOVA) showed genetic differences between and within populations (p = <0.01). Our data suggest that gene flow has occurred between the four populations of Tipularia discolor, although field studies have documented that populations are maintained through clonal growth. This study illustrates that ISSR markers can be used to detect genetic diversity at a population level.