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Spermatogonial stem cells (SSC) self-renew and differentiate into spermatozoa throughout the life of the male. SSC transplantation is a potential method for the propagation of genetically important males. These cells have been isolated in different mammalian species using specific cell surface markers, but not in felines. The goal of this study was to explore a relevant strategy for conservation of endangered felids by characterizing domestic cat (Felis catus) SSCs and assessing their ability for self-renewal after transplantation. Firstly, SSC and pluripotent surface markers, identified in non-feline species, were tested for expression in mixed germ cells from adults by immunocytochemistry and flow cytometry, with immunohistochemical confirmation of expression in prepubertal and adult testis tissue. Secondly, subpopulations were purified through fluorescence-activated cell sorting using spermatogonia-specific markers and molecularly characterized to ascertain levels of pluripotent transcription factors expressed in cat embryos. Thirdly, subpopulations of mixed germ cells and purified spermatogonial cells were transplanted to prepubertal cats to determine: 1) if SSCs capable of colonization were present, and 2) the value of using adolescent domestic cats without depletion of endogenous germ cells as recipients. Fourthly, various culture conditions were evaluated to identify proteins and factors required to maintain proliferation of cat SSCs. Lastly, adult lion testis tissue was characterized with the same surface markers, and mixed germ cells were transplanted to cat testes to evaluate the cat as a suitable host for lion SSC colonization and differentiation. Pluripotent surface markers were more reliable than the common SSC surface markers for isolating cat SSCs. Varying expression levels of pluripotent transcription factors between the different purified cell populations identified spermatogonial subpopulations. Cell purification was not necessary to colonize recipient testes, and transplantation validated the use of prepubertal males as recipients without depletion of endogenous cells. Unlike spermatogonia within mixed germ cells, purified spermatogonia were not maintained under various culture conditions; therefore, SSC culture conditions must be optimized. Similarities in the expression patterns of surface markers in lion and cat spermatogonia were revealed, and colonization of lion SSCs in cat testes provided further evidence of the domestic cat’s relevance as a model for exotic felid SSC transplantation.