Motility is an adaption that has occurred to allow organisms to explore new niches, escape from predation and to search for nutrients. Most bacteria swimming is powered by the bacterial flagellar motor which is comprised of many proteins, including flagellin, which is the major component of the flagellar filament. FliC is the protein which forms the filament in Escherichia coli and Salmonella enterica strains which contains of four domains, D0, D1, D2 and D3 (1, 2). The filament is made of approximately 30,000 flagellin monomers and is several micrometers in length and approximately 20 nm in diameter (3). The D0 and D1 domains are highly conserved across bacterial species, are formed of α-helices and are required for the formation of the helical core of the filament (4). Conversely, the D2 and D3 domains are made from highly variable β-sheets and in some cases are absent from the filament protein (4). Understanding the evolution of this protein and how it has diverged across the bacterial species is important to understand the origin of movement in nature and can provide key insights into manipulating motility through synthetic biology. In this work we have shown that the D2 and D3 domains have been gained and lost throughout evolutionary history. BLAST analysis of extant flagellin proteins from across the bacterial tree of life resulted in 224 representative flagellin amino acid sequences. Using these sequences, a MUSCLE alignment was performed after which a maximum likelihood phylogenetic tree was calculated using RAxML. We performed ancestral sequence reconstruction (ASR) to estimate residues sequences of the tree nodes and identified nine ancestral candidates from across the phylogenetic tree for further in vitro analysis. The phylogenetic analysis of the extant flagellin sequences provides evidence that the D2 and D3 domains were gained then subsequently lost through evolutionary history. Overall, our results show the minimal domains and specific residues within FliC which are required for rotary motility in bacteria. This study will provide a greater understanding about the evolution of the bacterial flagellar filament and the process of ASR to generate ancient flagellin candidates.