Atlantic halibut aquaculture would benefit from the development of all-female lines, since females grow faster and reach a larger size than males. Determining the period when gonad development occurs would define the critical time when hormonal or environmental treatments should be applied to influence sex ratios. To understand sex differentiation at the molecular level, we have characterized steroid hormone biosynthesis genes, including ovary- and brain-type aromatase and 11β-hydroxylase, which catalyze key steps in the synthesis of 17β-estradiol and 11-ketotestosterone, respectively. We have also investigated their expression during the period of sex differentiation in juveniles, as well as in various adult tissues, using Q-RT-PCR and have detected their expression in fish as small as 16 mm, which is earlier than when gonadal differentiation (GD) is detectable by histology (32 mm). In addition, partial cDNA sequences were obtained for steroid hormone receptor genes, steroidogenic enzyme genes including 17α-hydroxylase/C17, 21 lyase, 3β-hydroxysteroid dehydrogenase (HSD), 11β-HSD, 17β-HSD, and genes related to GD including three SRY-like box containing genes, sox-3, sox-5 and sox-9, anti-Mulllerian hormone, steroidogenic factor 1, Wilms’ tumor 1, and doublesex and mab-3 related transcription factor 1 (Dmrt1). We present the expression of these genes in various adult tissues and developing juveniles and relate them to aspects of sex specificity and GD. This study is a part of the Pleurogene project funded by Genome Canada/Genoma España.