Members of the Klotho gene family have been identified as modulators of the aging process. Deletion of αklotho in the mouse results in a syndrome resembling rapid human aging. Conversely, overexpression of αklotho extends mammalian lifespan. Here, we identify klotho orthologs in the vertebrate aging model Nothobranchius furzeri and provide a detailed spatio-temporal expression profile of both paralogs, α and βklotho, from embryogenesis until old age spanning the entire life cycle of the organism. Specifically, we observe low levels of expression of both paralogs during embryogenesis followed by a significant transcriptional induction as development proceeds. In adult killifish, αklotho is predominantly expressed in the liver, the kidney, and the developing pharyngeal teeth. Particularly high levels of αKlotho protein were identified in the kidney tubules, closely resembling mammalian expression patterns. Prominent βklotho expression was detected in the killifish intestine and liver. Overall, qRT-PCR analysis of Klotho members as a function of age revealed steady transcript levels, except for βklotho expression in the liver which was significantly downregulated with age. This spatio-temporal expression profiling may serve as a useful starting point to further investigate the distinct physiological roles of Klotho members during the aging process.