BACKGROUND: Diamondback moth Plutella xylostella (L.) (Lepidoptera: Plutellidae) is one of the most important crucifer pests. Commercial sex attractants have been developed to monitor and control P. xylostella. However, some studies have demonstrated a variety of pheromone responses of P. xylostella in different locations of the world. Soluble pheromone-binding proteins (PBPs), as a subfamily of odorant-binding proteins (OBPs), could selectively bind and transport pheromones across aqueous sensillar lymph to the surface of olfactory receptor neurons. It is worthy to study whether the mutation of PxylPBPs is one of the reasons for the different responses of sex attractors in different regions. RESULTS: In this study, P. xylostella males were collected from seven Chinese provinces, including Hainan, Guangdong, Yunnan, Fujian, Hunan, Zhejiang, and Hebei. PxylPBP1, PxylPBP2, and PxylPBP3 were cloned, and 3, 6, and 32 types of mutation pattern were identified, respectively. These mutation patterns were distributed in each province with different frequency. The results of fluorescence displacement binding assay and in silico simulation revealed that the three mutant PxylPBP3 were more sensitive to Z11-16:Ald than the reference protein (ACI28451). CONCLUSION: This result implied that mutation of PxylPBP3 may have contributed to regional differences in pheromone responses of P. xylostella.