The adaptation to novel host plants is an important factor that facilitates spread of tephritid fruit flies and formation of new biotypes. Bactrocera tau (Walker) (Diptera: Tephritidae) is a significant pest and member of the Tephritidae family that has expanded its normal host range from Cucurbitaceae to many other plant families. The objective of this study was to monitor the impact of novel host plants on genetic variation in B. tau. In this study, under laboratory conditions, we examined genetic variation based on microsatellite molecular markers of B. tau when development occurred on one novel host (banana) and two traditional hosts (pumpkin and cucumber) for 15 continuous generations. Analysis of molecular variance showed that the genetic difference (10.39%) among populations feeding on the various hosts was significant, but not among populations from different generations. It appears that host food, more than population generations, played a critical role in genetic variation of B. tau. The effect of hosts on genetic variation of B. tau was greater on the novel host compared with the traditional Cucurbitaceae hosts. The significant genetic variation of the banana-feeding populations was reflected in difficulties of balancing in frequency of gene and genotype, reduced genetic diversity, and more divergent genetic difference.