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ABSTRACT The prevalence and lineages of Streptococcus agalactiae (group B streptococci [GBS]) colonizing pregnant women are well studied, but less is known about colonization of nonpregnant adults. We characterized GBS colonization in adults as a potential reservoir for infections and tested for the presence of clones with a potentially higher invasive disease potential. We evaluated GBS gastrointestinal, genitourinary, and oral colonization among 336 nonpregnant adults in the community. We characterized the isolates by serotyping, multilocus sequence typing, profiling of surface protein genes and pili, and antimicrobial susceptibility and compared them with contemporary invasive isolates. The colonization rate (n = 107, 32%) among nonpregnant adults was like that of pregnant women. Colonization increased with age (~25% in the 18 to 29 and 30 to 44 years old groups and >42% in the ≥60 years old group), potentially explaining the higher incidence of disease with older age. Participants who were colonized at multiple sites (73%) were frequently carrying indistinguishable strains (93%), consistent with the existence of a single reservoir of colonization and transfer of GBS between sites within the same individual. The most frequent lineages found were serotype Ib/CC1 (n = 27), serotype V/CC1 (n = 19), serotype Ia/CC23 (n = 13), serotype III/ST17 (n = 13), and serotype Ib/CC10 (n = 11). Comparison with contemporary isolates causing invasive infections in Portugal did not reveal any lineage associated with either asymptomatic carriage or invasive disease. Asymptomatic colonization of nonpregnant adults is significant and could act as a reservoir for invasive disease, but in contrast to infant disease, we found no GBS lineages with an enhanced potential for causing invasive disease in adults. IMPORTANCE The increasing incidence of Streptococcus agalactiae (group B streptococci [GBS]) infections in adults and the inability of antimicrobial prophylaxis peripartum to control late-onset infections in infants motivate the study of the asymptomatic carrier state in nonpregnant adults. We found an overall carriage rate like that of pregnant women, increasing with age, potentially contributing to the higher incidence of GBS infections with age. Colonization of diabetic participants was not higher despite the higher number of infections in this group. Comparison between contemporary genetic lineages causing infections and found in asymptomatic carriers did not identify particularly virulent lineages. This means that any prophylactic approaches targeting colonization by particular lineages are expected to have a limited impact on GBS disease in adults.