Abstract Bemisia tabaci (Gennadius) is an important agricultural pest with a worldwide distribution. Although B. tabaci is known to have a unique haplodiploid reproductive strategy, its sex determination mechanism is largely unknown. In this study, we cloned the full‐length sequence of B. tabaci doublesex ( Btdsx ) and found that Btdsx has 28 splicing isoforms. We found two new splicing isoforms of transformer 2 ( Bttra2 ), which encode two proteins. We also confirmed that both genes lack sex‐specific splicing isoforms. Real‐time quantitative PCR analysis showed that the expression of Btdsx and Bttra2 is higher in males than in females. RNA interference of Bttra2 affected the expression of Btdsx and vice versa. Furthermore, silencing of Bttra2 or Btdsx caused malformation of the male genitalia (anal style). It did not affect the female phenotype, but reduced the expression of vitellogenin gene in females. These results indicate that Btdsx is associated with sex determination in B. tabaci and that Btdsx and Bttra2 affect each other and are important for male genitalia formation. In addition to increasing our understanding of the roles of dsx and tra2 in the sex determination of B. tabaci , the results will be useful for studies of sex determination in other haplodiploid species.