<p>The role of gut microbiota in avian health is well understood, yet the process by which it assembles in neonatal penguins, a species that acts as a sentinel in polar environments, remains poorly understood. In this study, we used high-throughput 16&#xa0;S rRNA gene sequencing to analyse the dynamics and assembly of the gut microbiota of neonatal gentoo penguins in a captive population. While living in the nest with their parents, the gut microbiota of the neonates had a similar community structure to the eggshell microbiome in terms of diversity, composition and function. However, once they left the nest, there was significant variation in the gut microbiota of the chicks and the individual differences increased. Specifically, diversity decreased, inferred potential functions were reduced and the abundance of the bacterial genus <i>Paeniclostridium</i> increased in the gut microbiota of penguin chicks after they left the nest. Heterogeneous selection completely controlled the assembly of the eggshell microbiome when the egg was first laid; then, homogenizing dispersal played an important role. Additionally, the role of homogenizing dispersal in shaping the gut microbiota of penguin chicks diminished as they grew. After leaving the nest, stochastic drift partially controlled the assembly of the community. Source tracking analysis estimated that approximately half of the gut microbiota in newborn penguins originated from the eggshell microbiome. This proportion decreased as the chicks grew, while microbes derived from material transmission increased steadily, especially after they left the nest. These results provide new insights into the assembly and sources of gut microbiota in neonatal penguins, offering important clues for improving the health of captive penguins through microbiome-based strategies.</p>