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Frozen\u2010thawed semen was inseminated into five anesthetized females, over a total of eight egg production cycles, with a different male used for each artificial insemination (AI) within each season. Females were maintained within the colony in cordoned nest sites to prevent copulation with their paired male, and were inseminated every 3\u201310 days until the first oviposition. Semen frozen from seven males using a straw method retained 39.8%, 25.7%, 74.0%, and 52.1% of its initial total motility, progressive motility, average path velocity, and plasma membrane integrity, respectively. Normal morphology of motile cells was reduced (<jats:italic>P<\/jats:italic>\u2009&lt;\u20090.05) during freeze\u2010thawing from 76.7% immediately prior to freezing to 65.3% post\u2010thawing. Conceptive females received 1.6\u2009\u00b1\u20090.2 inseminations before the first oviposition, with 19.2\u2009\u00b1\u20091.6\u2009\u00d7\u200910<jats:sup>6<\/jats:sup> motile, morphologically normal spermatozoa per insemination. Overall fertility was 53.3% (8\/15 eggs), hatchability was 50.0% (4\/8), and genetic analyses confirmed that all embryos and hatchlings were sired by the AI male. Fertile eggs were laid at 4.0\u201312.1 days after AI, indicating that frozen\u2010thawed spermatozoa resided in the female reproductive tract for up to \u223c7.2\u2009days prior to fertilization. Results demonstrate that frozen\u2010thawed Magellanic penguin spermatozoa are fully functional in vivo and support the use of genome banking and AI as tools for managing the sustainability of zoological penguin populations. 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