Microsatellite and mitochondrial DNA (mtDNA) variability data were used to study infestations of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann) in Florida in 1997 and 1998. A total of 132 flies collected in monitoring traps or as larvae removed from fruit were examined at three polymorphic mtDNA restriction sites and two microsatellite loci. All of the flies sampled in Florida in 1997 displayed the mitochondrial AAB haplotype and represent a novel introduction of Mediterranean fruit flies into the state. All flies collected in central Florida in 1998 also displayed the AAB haplotype. Microsatellite analysis of these specimens from 1998 detected only alleles that were present in 1997. These results strongly indicate that the 1998 Florida outbreaks were derived from the Florida populations from the previous year. According to our analyses, the Mediterranean region is the most likely source for the 1997 Florida infestation. Flies from a small outbreak in Miami Springs, Dade County, FL, early in 1998 had a different mtDNA haplotype, characterized by the AAC restriction pattern. Microsatellites of these specimens showed significant differences in their allelic distribution from AAB flies, indicating an origin from a separate source population. South America is the most likely source for the Miami Springs flies.