The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is one of the most serious pests for cruciferous vegetable growers worldwide. To determine the relations of local and regional populations, we performed a mitochondrial COI gene analysis of eight P. xylostella populations from different locations in or around the Qinling Mountains and two other populations, one from Beijing and the other from Guangdong. The mtDNA divergences among the 10 populations were high, and 32 haplotypes were detected in 149 adults. The mean haplotype divergence was 1.7% (range 0.04–4.1%). Haplotype diversity in the 10 populations varied from 0.571 (AK) to 0.885 (HZ), and the nucleotide diversity varied from 0.00286 (AK) to 0.0117 (HZ). The results also did not show significant correlation between genetic and geographical distance. Also, the effective number of migrants between populations (Nm) ranged from 1.43 to infinite, suggesting that population exchange and gene flow among the P. xylostella populations occurred. However, principal component analysis (PCA) showed that the TB and TC populations were differentiated from other populations, indicating possible across-mountain barrier to migration and gene flow.