Understanding the relationship between incursions of insect pests and established populations is critical to implementing effective control. Studies of genetic variation can provide powerful tools to examine potential invasion pathways and longevity of individual pest outbreaks. The major fruit fly pest in eastern Australia, Queensland fruit fly Bactrocera tryoni (Froggatt), has been subject to significant long-term quarantine and population reduction control measures in the major horticulture production areas of southeastern Australia, at the species southern range limit. Previous studies have employed microsatellite markers to estimate gene flow between populations across this region. In this study, we used an independent genetic marker, mitochondrial DNA (mtDNA) sequences, to screen genetic variation in established and adjacent outbreak populations in southeastern Australia. During the study period, favorable environmental conditions resulted in multiple outbreaks, which appeared genetically distinctive and relatively geographically localized, implying minimal dispersal between simultaneous outbreaks. Populations in established regions were found to occur over much larger areas. Screening mtDNA (female) lineages proved to be an effective alternative genetic tool to assist in understanding fruit fly population dynamics and provide another possible molecular method that could now be employed for better understanding of the ecology and evolution of this and other pest species.