Simultaneous hermaphrodites, which reproduce reciprocally, are good model organisms to study evolution of sex determination. First of all, acquisition of a mutation causing sterility in one sex role (i.e. a sex determining gene) can lead to selection favouring linked sex-specific fitness alleles. This is expected to be followed by recombination suppression between these loci, leading to the evolution of sex chromosomes. Later acquirement of an opposite sex role sterility mutation in the hermaphroditic partner leads to the final evolution of two separate sexes. The non-recombining sex-determining region later expands by acquiring more sex-specific genes, thus causing sex chromosomes to become heteromorphic. However, as both sexes still share most of the genome, shared genes with opposite fitness effects between the sexes must acquire sex-biased or sex-specific gene expression. Thus, to experimentally observe this process, we subjected a simultaneous reciprocally mating hermaphrodite Macrostomum lignano to sex-limited selection using a GFP marker as a sex-determining locus. In this setup male-limited selection resembles XY chromosome evolution, while female-limited selection – ZW. After 21-22 generations of sex-limited experimental evolution, we performed RNA-seq to see if we can indeed observe divergence in expression profiles of male- and female-selected lines. We hope that our results will show that there is an evolving divergence between male- and female-selected lines compared to controls. The experimental evolution is still ongoing, so since we know that organ size affects sex-specific fitness and the lines have changed somewhat in organ size, we also plan to investigate organ-specific expression in the future.