Populations of Rhagoletis mendax with novel late flight period, in August–September, have been found in two commercial highbush blueberry fields in New Jersey, thriving in fruit left behind after the harvest of a late blueberry cultivar. A small number of flies with similar late flight period have also been captured in several locations where blueberry maggot infests wild hosts. Using allozyme markers, we found that allele frequencies in wild blueberry maggot populations are constant from June to September, and distinct from those found in populations infesting the two commercial fields. The populations in the commercial fields present a pattern of decreased genetic diversity, including lower heterozygosity and distortions in allele frequency distribution. These data suggest that a founder effect, the loss of genetic diversity caused by the establishment of a small sample of individuals as a newly isolated entity, is responsible for the genetic differences between the populations in the commercial fields and in wild areas. The founder effect most likely occurred because only a small proportion of the flies present in wild areas possesses the late phenology required for the colonization of the commercial fields. The seasonal asynchrony between the populations in commercial fields and those in wild areas may also have contributed to maintain the founder effect by limiting gene flow. This study suggests that early in a phenological transition, if the onset of prezygotic reproductive isolation is relatively fast, evolution at the genetic level might be caused mostly by genetic drift. This study further confirms that the flight period is plastic in the blueberry maggot.