Sex determination genes are important regulators of reproduction as well as of the development of both behavioural and morphological sex characteristics. The sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae), is an emerging insect pest worldwide. The recent release of the genome sequence of the highly invasive B. tabaciQ/MED allows us to investigate the mechanisms and genes involved in sex determination. The combined genome and transcriptome-wide analyses identified 26 genes putatively associated with sex determination in B. tabaciQ/MED. The temporal profiles of these genes exhibited a consistent expression pattern at all B. tabaci developmental stages: the highest transcript levels were detected in eggs (21 genes, 80.8%) and the lowest in adults (24 genes, 92.3%). The expression pattern was further validated by quantitative real-time reverse transcription PCR analysis. Alternative splicing analysis found that (1) da and mle have sex-specific isoforms in B. tabaci adults, whereas Imp does not, and (2) exon skipping is a common splicing mechanism involved in B. tabaci sex determination. This research provides a comprehensive list of genes involved in B. tabaci sex determination and provides an opportunity to further understand the mechanisms underlying sex determination in a globally invasive insect pest that reproduces both sexually and asexually. © 2019 The Royal Entomological Society.