Wolbachia pipientis is an obligate, intracellular Alphaproteobacterium, related to Rickettsia. Itis the most common endosymbiont of insects and 40% of all arthropods are estimated to be infected with Wolbachia. The transmission of Wolbachia is mainly vertical by maternal inheritance, while its manipulation of host reproductive systems can increase the number of infected females in host populations. For example, Wolbachia can bias host sex ratios and modify host sex determination through cytoplasmic incompatibility, parthenogenesis, male-killing or feminisation. Feminisation is the least reported phenotype, mostly detected in terrestrial crustaceans. It also occurs in three insect species, including two closely related butterfly species in the genus Eurema. Feminisation cause genetic males to develop into phenotypic females. In butterflies, the detection of the W chromatin body in females has so far been the main evidence for feminisation. However, this method is not reliable for all Lepidoptera and a molecular method based on sex chromosomal markers may be better. We developed a quantitative PCR (qPCR) assay, comparing relative gene dose ratios of Z-chromosomal genes with an autosomal gene, to differentiate directly between true genotypic females and Wolbachia feminised males. This test correctly genotyped the sex of butterflies that lacked the feminising Wolbachia strain wFem. However, all wFem infected females that were negative in the W chromatin body assay (thus considered feminised males) also had female gene dose ratios. These results conflict with the current model of feminisation of males in Eurema and suggest re-assessment of the role of Wolbachia as feminising agent in this host.