Genetic relationships were examined among wild-caught southern flying squirrels (Glaucomys volans) sharing the same natural nest cavity. Under natural conditions, typically 75–80% of southern flying squirrel nest groups comprise adult-aged individuals. The remainder nest in family-based groups or are solitary. The coefficient of relatedness within nest groups of adult individuals and family-based nest groups was examined through microsatellite DNA analysis. Family or adult nest groups were identified from the age class of individual group members determined through a discriminant function analysis based on body mass. From this information, nest groups were categorized as family-based groups comprising a single adult female with nestlings, adult groups comprising adult aged-individuals, or subadult nest groups. The average coefficient of relatedness was determined in each nest group. Within the putative family groups, most individuals were 1st-order relatives. In the adult nest groups, the coefficient of relatedness was low, indicating that these individuals were unrelated. The relationships within the subadult nest groups were intermediate. This is the 1st study to show that adult nestmate southern flying squirrels typically are unrelated and do not nest in family-based groups.