Viburnum rufidulum is a deciduous tree native to North America that has four-season appeal, which provides commercial horticultural value. In addition, the plant has unique and attractive red pubescence on leaf buds and petioles, common to no other Viburnum species. As habitat undergoes development and subsequent fragmentation of native plant populations, it is important to have baseline genetic information for this species. Little is known about the genetic diversity within populations of V. rufidulum. In this study, seven microsatellite loci were used to measure genetic diversity, population structure, and gene flow of 235 V. rufidulum trees collected from 17 locations in Kentucky and Tennessee. The genotype data were used to infer population genetic structure using the program InStruct and to construct an unweighted pair group method with arithmetic mean dendrogram. A single population was indicated by the program InStruct and the dendrogram clustered the locations into two groups; however, little bootstrap support was evident. Observed and expected heterozygosity were 0.49 and 0.78, respectively. Low-to-moderate genetic differentiation (FST = 0.06) with evidence of gene flow (Nm = 4.82) was observed among 17 populations of V. rufidulum. A significant level of genetic diversity was evident among V. rufidulum populations with most of the genetic variations among individual trees (86.37%) rather than among populations (13.63%), and a Mantel test revealed significant correlation between genetic and geographical distance (r = 0.091, P = 0.001). The microsatellites developed herein provide an initial assessment or a baseline of genetic diversity for V. rufidulum in a limited area of the southeastern region of the United States. The markers are a genetic resource and can be of assistance in breeding programs, germplasm assessment, and future studies of V. rufidulum populations, as this is the first study to provide genetic diversity data for this native species.