Little is known about the genetic basis of sex determination in vertebrates though considerable progress has been made in recent years. In this study, segregation analysis and linkage mapping were performed to localize an amphibian sex-determining locus (ambysex) in the tiger salamander (Ambystoma) genome. Segregation of sex phenotypes (male and female) among the second generation individuals of interspecific crosses (Ambystoma mexicanum x Ambystoma tigrinum tigrinum) was consistent with Mendelian expectations, although a slight female bias was observed. Individuals from these same crosses were typed for single-nucleotide polymorphisms distributed throughout the genome to identify molecular markers for ambysex. A marker (E24C3) was identified approximately 5.9 cM from ambysex. Linkage of E24C3 to ambysex was independently validated in a second, intraspecific cross (A. mexicanum). Interestingly, ambysex locates to the tip of one of the larger linkage groups of the Ambystoma meiotic map. Considering that this location does not show reduced recombination, we speculate that the ambysex locus may have arisen quite recently, within the last few million years. Localization of ambysex sets the stage for gene identification and provides important tools for studying the effect of sex in laboratory and natural populations of this model amphibian system.