The fungus gnat, Bradysia difformis (Sciaridae: Diptera) has recently been recorded for the first time from South Africa where it has been found in forestry nurseries. The presence of this insect in all the major forestry nurseries as the dominant and only sciarid species raises intriguing questions regarding its origin and population genetic structure. A 395 bp portion of the mitochondrial COI gene was analysed from B. difformis individuals collected from four nursery populations in South Africa and three nursery populations in Europe. Shared haplotypes between South African and European populations indicated a historical connection. South African populations showed high genetic diversity and low genetic differentiation. These patterns most likely reflect multiple and/or relatively large introductions of B. difformis into South Africa from its origin combined with subsequent and continued movement of plants between nurseries.