The PsbO protein is a ubiquitous extrinsic subunit of Photosystem II (PS II), the water splitting enzyme of photosynthesis. A recently determined 3D X-ray structure of a cyanobacterial protein bound to PS II has given an opportunity to conduct complete analyses of its sequence and structural characteristics using bioinformatic methods. Multiple sequence alignments for the PsbO family are constructed and correlated with the cyanobacterial structure. We identify the most conserved regions of PsbO and the mapping of their positions within the structure indicates their functional roles especially in relation to interactions of this protein with the lumenal surface of PS II. Homologous models for eukaryotic PsbO were built in order to compare with the prokaryotic protein. We also explore structural homology between PsbO and other proteins for which 3D structures are known and determine its structural classification. These analyses contribute to the understanding of the function and evolutionary origin of the PS II manganese stabilising protein.