Streptococcus pneumoniae strains are the leading cause of bacterial otitis media, yet little is known about specific bacterial factors important for this disease. We utilized a molecular epidemiological approach involving genomic subtraction of the S. pneumoniae serogroup 19 middle ear strain 5093 against the laboratory strain R6. Resulting subtraction PCR (sPCR) products were used to screen a panel of 93 middle ear, 90 blood, 35 carriage, and 58 cerebrospinal fluid isolates from young children to identify genes found more frequently among middle ear isolates. Probe P41, similar to a hypothetical protein of Brucella melitensis, occurred among 41% of middle ear isolates and was found 2.8 (95% confidence interval [CI], 1.32 to 6.5), 3.3 (95% CI, 1.9 to 5.7), and 1.8 (95% CI, 1.1 to 3.0) times more frequently among middle ear strains than carriage, blood, or meningitis strains, respectively. sPCR fragment H10, similar to an unknown Streptococcus agalactiae protein, was present in 31% of middle ear isolates and occurred 3.6 (95% CI, 1.2 to 11.2), 2.8 (95% CI, 1.5 to 5.4), and 2.6 (95% CI, 1.2 to 5.5) times more often among middle ear isolates than carriage, blood, or meningitis strains, respectively. These studies have identified two genes of potential importance in otitis media virulence. Further studies are warranted to outline the precise role of these genes in otitis media pathogenesis.