The introduction of the seven-valent pneumococcal conjugate vaccine (PCV7) in Portugal led to extensive serotype replacement among carriers of pneumococci, with a marked decrease of PCV7 types. Although antimicrobial resistance was traditionally associated with PCV7 types, no significant changes in the rates of nonsusceptibility to penicillin, resistance to macrolides, or multidrug resistance were observed. This study aimed to investigate the mechanisms leading to maintenance of antimicrobial resistance, despite marked serotype replacement. We compared, through molecular typing, 252 antibiotic-resistant pneumococci recovered from young carriers in 2006 and 2007 (era of high PCV7 uptake) with collections of isolates from 2002 and 2003 (n ؍ 374; low-PCV7-uptake era) and 1996 to 2001 (n ؍ 805; pre-PCV7 era). We observed that the group of clones that has accounted for antimicrobial resistance since 1996 is essentially the same as the one identified in the PCV7 era. The relative proportions of such clones have, however, evolved substantially overtime. Notably, widespread use of PCV7 led to an expansion of two Pneumococcal Molecular Epidemiology Network (PMEN) clones expressing non-PCV7 capsular variants of the original strains: Sweden 15A ST63 (serotypes 15A and 19A) and Denmark 14 ST230 (serotypes 19A and 24F). These variants were already in circulation in the pre-PCV7 era, although they have now become increasingly abundant. Emergence of novel clones and de novo acquisition of resistance contributed little to the observed scenario. No evidence of capsular switch events occurring after PCV7 introduction was found. In the era of PCVs, antimicrobial resistance remains a problem among the carried pneumococci. Continuous surveillance is warranted to evaluate serotype and clonal shifts leading to maintenance of antimicrobial resistance.Streptococcus pneumoniae is a Gram-positive bacterium that frequently colonizes asymptomatically the nasopharynx of young children. However, it is also an important human pathogen that can cause a wide range of diseases, from otitis media to pneumonia and meningitis. Worldwide, it has been estimated that 14.5 million episodes of serious pneumococcal disease and 826,000 deaths per year occur in children aged less than 5 years (23).In 2000, a seven-valent pneumococcal conjugate vaccine (PCV7) targeting the seven most common serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F) causing invasive disease among young children in the United States became available. Since then, in countries where the vaccine has been introduced in the universal vaccination plan, a dramatic reduction in the incidence of invasive disease caused by PCV7 serotypes has been observed in all age groups (11,17,35). This extended phenomenon beyond the target group, known as herd immunity, has been attributed to decreased transmission of pneumococci from children to other age groups. Indeed, the nasopharynx of children is the main reservoir of pneumococci, and a decrease in carriage of PCV7 serotypes has been observed in vaccina...