Serotype 1 pneumococci are rarely isolated from carriers, but are an important cause of pneumococcal invasive disease in many regions of the world. This report describes the emergence and expansion of a single serotype 1 lineage (characterised by multilocus sequence type 306) among healthy carriers attending day-care centres in Portugal. The prevalence of serotype 1 strains among all pneumococci increased from 0% in 2001 and 2002, to 0.4% in 2003, and 3.1% in 2006. These observations paralleled the introduction and increased use of the seven-valent pneumococcal conjugate vaccine in the study group, suggesting a direct relationship between these events.