Digitonthophagus gazella (F.) has been widely introduced to the New World and both natural dispersal and intentional releases continue. In this study, we compare a population of D. gazella from South Africa and from the island of Vieques, Puerto Rico, using amplified fragment length polymorphism (AFLP) analysis. Genetic diversity was found to be high in both South Africa (H = 0.3623) and Vieques (H = 0.3846), providing no evidence of inbreeding depression on Vieques. Analysis of molecular variance (AMOVA) revealed that 69% of genetic diversity is within the populations and 31% of genetic diversity is between the populations, indicating that if interbreeding occurs between these populations, it is rare and likely human-mediated. The Fst value of 0.3143 also suggests that there is genetic isolation between populations in Africa and newly established populations in the New World. Because of its competitiveness and natural dispersal ability, additional comparisons of D. gazella populations, biology, and genetics are warranted.