Sequence and length-polymorphic intron variations in the caboxylesterase 2 gene (coe2) were determined to be specific to the biotypes B and Q of Bemisia tabaci. By employing the biotype-specific coe2 intron variation as a nuclear marker, a one-step diagnostic protocol for the identification of B and Q biotypes was developed and its performance was validated for field collected B. tabaci specimens. The diagnostic results based on the coe2 intron marker were identical to those obtained from the mtCOI marker in all 256 specimens examined except for four individuals that appeared to be putative heterozygotes between B and Q biotypes. These results demonstrate a high level of accuracy of the coe2 intron marker-based protocol in distinguishing biotypes B and Q. Moreover, because the process requires only PCR amplification and gel electrophoresis, analysis of multiple samples can be done more efficiently. Based on the observation that all putative heterozygotes have the maternal background of Q biotype, they may have been created by inter-biotype cross between B type male and Q type female. If combined with the mtCOI marker, the nuclear coe2 marker would provide a better resolution than maternally inherited markers alone and facilitate the demographic study of B. tabaci biotype complex.