Many taxa have independently evolved genetic sex determination where a single gene located on a sex chromosome controls gonadal differentiation. The gene anti-Mullerian hormone (amh) has convergently evolved as a sex determination gene in numerous vertebrate species, but how this gene has repeatedly evolved this novel function is not well understood. In the threespine stickleback (Gasterosteus aculeatus), amh was duplicated onto the Y chromosome (amhy) ~22 million years ago. To determine whether amhy is the primary sex determination gene, we used CRISPR/Cas9 and transgenesis to show that amhy is necessary and sufficient for male sex determination, consistent with the function of a primary sex determination gene. Despite being indispensable for sex determination, we detected low levels of amhy expression throughout early development. This indicates the mechanism of sex determination is likely unrelated to overall dosage of amhy and its autosomal paralog, amha. Threespine stickleback have striking differences in behavior and morphology between sexes. The creation of sex reversed lines allow us to investigate the genetic basis of secondary sex characteristics. Here we show one of the classic traits important for reproductive success, male nuptial coloration, is controlled by both Y-linked genetic factors as well as hormonal factors independent of sex chromosome genotype. This research establishes stickleback as a model to investigate how amh regulates gonadal development and how this gene repeatedly evolves novel function in sex determination. Analogous to the four core genotypes model in house mice, sex-reversed threespine stickleback offer a new vertebrate model for investigating the separate contributions of gonadal sex and sex chromosomes to sexual dimorphism.