Exogenous treatment of fish with natural sex hormones and their mimics has been shown to influence gonadal differentiation resulting in biased phenotypic sex-ratios. This has lead to the development of the Fish Sexual Development Test (FSDT) as a method for the detection of endocrine active chemicals. Proposed test organisms include the medaka, zebrafish (ZF) and stickleback, although the guideline also allows for inclusion of species such as the fathead minnow (FHM), provided the test duration allows for sufficient sexual differentiation. However, although the processes underlying sexual differentiation are known to differ for each of these species, it is not known how, or if, these differences would influence the results of the FSDT. In the experiments reported here, responses of the ZF and FHM to prochloraz, a sterol biosynthesis inhibitor and androgen antagonist, were characterized and compared. Exposure to 320 μg/L of prochloraz, from embryo until 60 (ZF) or 95–125 (FHM) days post hatch inhibited somatic growth of both species, but while a negative impact on ZF larval survival was observed (LOEC 32 μg/L) there was no evidence for an effect on FHM larval survival. Prochloraz influenced sexual differentiation in both species by decreasing the proportion of females (LOEC 100 μg/L (ZF), 320 μg/L (FHM)) and delaying completion of sexual differentiation; manifest as an increased incidence of ovotestis in the ZF (LOEC 100 μg/L) and as an increased number of fish with undifferentiated gonads in the FHM (LOEC 320 μg/L). However, while exposure to 320 μg/L prochloraz delayed maturation of the differentiated FHM testis, there was no such effect in the ZF. These results demonstrate that the different strategy of sexual differentiation in the ZF and FHM influences the profile of responses of their gonads to the masculinising effects of prochloraz, but does not affect their overall sensitivity.