Streptococcus pneumoniae (the pneumococcus) is one of the most common bacterial causes of middle ear infections (otitis media) in children. These infections often resolve without intervention, but can sometimes be persistent and challenging to treat. S. pneumoniae is highly genetically diverse and while the molecular factors that influence disease are not fully understood, bacterial metabolic capacity is known to play an important role in infection. Previous studies in our laboratory have demonstrated that pneumococcal isolates taken from the ear displayed polymorphisms in genes encoding the uptake and regulatory pathways for raffinose-family oligosaccharides (rafK and rafR), which were associated with reduced raffinose metabolism in vitro and had a marked influence on disease tropism in animal models of infection. In the present study, we assessed eight isolates of S. pneumoniae serotype 3 taken from the middle ear of children with otitis media, and showed one was unable to metabolise raffinose. Serotype 3 is a clinically relevant serotype in otitis media and is a serotype of concern associated with significant vaccine escape. We next performed whole genome sequencing and variant analyses to identify potential molecular factors associated with the observed metabolic defect. These studies identified a premature stop codon in the gene encoding the raffinose ABC transporter substrate-binding protein (rafE). Together, this work provides further evidence supporting potential variation at the pneumococcal raffinose utilisation locus in pneumococci isolated during an episode of otitis media, which aids our understanding of these infections and may have implications for the design of new therapies and vaccines.