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The Bemisia tabaci Mediterranean (MED) cryptic species is an invasive pest, distributed worldwide, with high ecological adaptability and thermotolerance. DNA methylation (a reversible chromatin modification) is one possible change that may occur within an organism subjected to environmental stress. To assess the effects of temperature stress on DNA methyltransferase 3 (Dnmt3) in MED, we cloned and sequenced BtDnmt3 and identified its functions in response to high and low temperatures. The full‐length cDNA of BtDnmt3 was 3913 bp, with an open reading frame of 1962 bp, encoding a 73.89 kDa protein. In situ hybridization showed that BtDnmt3 was expressed mainly in the posterior region. BtDnmt3 messenger RNA expression levels were significantly down‐regulated after exposure to heat shock and significantly up‐regulated after exposure to cold shock. Furthermore, after feeding on double‐stranded RNA specific for BtDnmt3, both heat resistance and cold resistance were significantly decreased, suggesting that BtDnmt3 is associated with thermal stress response and indicating a differential response to high‐ and low‐temperature stress in MED. Together, these results highlight a potential role for DNA methylation in thermal resistance, which is a process important to successful invasion and colonization of an alien species in various environments.