Flea beetles and cabbage butterfly (Pieris rapae) are the most destructive and devastating pests of cruciferous crops worldwide. Individually, they may cause yield losses as much as 100% in brassicas and therefore cripple the economy of smallholder farmers especially in Southeast Asia. In order to understand the genetic diversity, phylogeography and intraspecific genetic variation among these pest populations, mitochondrial cytochrome c oxidase 1 (mtcox1) gene was used. We first explored 89 individuals of Pieris and 187 individuals of flea beetle populations from five different geographically distinct regions including Taiwan, Cambodia, Vietnam, Thailand and Laos. Individual haplotype diversities based on cox1 gene showed 10 and 35 different haplotypes of Pieris and flea beetles, respectively. Furthermore, Tajima’s D, Fu’s Fs and Analysis of Molecular Variance (AMOVA) of pairwise comparison (Fst) tests were performed across all Pieris and flea beetle populations in target countries. The results showed low nucleotide diversity, high genetic differentiation and gene flow, which suggest recent population expansion especially between Taiwan and Thailand populations of flea beetles. Similarly, isolated populations of P. rapae have higher haplotype diversity (h>28) and lower nucleotide diversity (0.00033) in Vietnam and Thailand. In addition, genetic differentiation results showed that the Taiwan population could be a separate species. Thus, our results provided better understanding on the genetic diversity of flea beetle and cabbage butterfly populations in Southeast Asia.