Bemisia tabaci (Gennadius) is a worldwide pest of vegetable, ornamental and field crops. Biotype B of B. tabaci, which is economically most important of the biotypes, is distinct from all other biotypes (non-B biotypes). Fourteen populations of B. tabaci were collected from different localities and host plants in the Chinese mainland and Taiwan, namely TWYDH (tassel flower, Taiwan), HNYC (tobacco, Hainan), GXNG (pumpkin, Guangxi), GDYPH (poinsettia, Guangdong), GDBSM (croton, Guangdong), GDFS (Chinese hibiscus, Guangdong), SHYPH (poinsettia, Shanghai), FJGS (sweet potato, Fujian), SDFQ (tomato, Shandong), BJXHL (squash, Beijing), XJQZ (eggplant, Xinjiang), XJYPH (poinsettia, Xinjiang), XJJM (abutilon, Xinjiang) and XJMH (cotton, Xinjiang). The internally transcribed spacer 1 sequences (ITS1) of ribosomal DNA of B biotype and other biotypes were sequenced and analyzed. The B biotype-specific primers were then designed for rapid identification of B biotype of B. tabaci. The results show that the diagnostic primer only gave a positive result with the B biotype. This is the first report of a rapid means of identifying B. tabaci B biotype using a diagnostic primer based on ribosomal DNA. This protocol is especially useful for identifying the B biotype in Bemisia populations consisting of several biotypes.