The zebrafish (ZF) undergoes juvenile hermaphroditism, where in all fish, the gonad first develops as an ovary-like structure, before the sex is fixed and the gonad then differentiates into either a testis or an ovary. Sexual differentiation is being adopted as an endpoint for assessing endocrine disrupting chemicals, yet there is only very limited information on the timing and variability in sexual differentiation process both within and between strains of ZF. In this study, using gonad histology, the ontogeny of sexual differentiation was studied in two strains of ZF, one with a high level of heterozygocity (WIK strain) and another that had been in-bred for several generations. There was a high variability in the timing of sex differentiation between individuals within a specific strain (with no obvious relationship with body mass), but there were no differences between the two strains. Transformation of the immature gonad into testes in males started in week 5 post fertilization (pf) and completion of sexual differentiation for all fish in both study populations occurred by week 11–12 pf. The size of the fish containing transforming gonads in the WIK strain and in-bred strain were similar (ranging from 12 to 23 mm, and from 13 to 22 mm total length, respectively).