this study aimed to characterise the GI microbiota of little penguin chicks from birth to fledging (56-70 days), using Quantitative Real Time PCR (qPCR) and 16S rRNA pyrosequencing sequencing. Results from the qPCR analysis showed an upward trend for Firmicutes and Bacteroidetes during developing in the little penguin chick. No trend was observed for Proteobacteria or Actinobacteria. 16S rRNA pyrosequencing data identified that there are significant fluctuations in the microbial composition of little penguins from hatching to fledging. The similarity between all age classes is around 20%, with similarity slightly increasing to 40% between weeks 5, 7 and 9. The newly hatched chick is quickly colonised by aerobic and facultative anaerobic bacteria, with Enterobacteriaceae dominating the microbiota throughout the first three weeks of development. By week 5, the composition is dominated by Fusobacteriaceae and Clostridiaceae, with Fusobacteriaceae and Clostridiaceae continue to dominate throughout development until fledging. Similarly to other vertebrates, there is initial increase in species diversity, followed by a steady decline by fledging. This study describes the first molecular examination of the microbial community of the digestive tract of little penguin chicks throughout development. Little penguin chicks display similar ecological successional changes as other vertebrates with aerobic and facultative anaerobes being the first microbes to colonise the GI tract and slowly transitioning to a microbiota dominated by strict anaerobes. However unlike other vertebrate species, the little penguin microbiota at fledging is still relatively unstable and does not resemble that of the adult microbiota