Abstract Background The substantial risk for respiratory and invasive infections with Streptococcus pneumoniae (Spn) among people with HIV-1 (PWH) begins with asymptomatic colonization. The frequency of Spn colonization among US adults with and without HIV-1 infection is not well characterized in the conjugate vaccine era. Methods We determined Spn colonization frequency by culture and specific lytA gene quantitative polymerase chain reaction (PCR) and microbiota profile by 16S ribosomal RNA gene sequencing in nasopharyngeal (NP) and oropharyngeal (OP) DNA from 138 PWH and 93 control adults and associated clinical characteristics. Results The frequencies of Spn colonization among PWH and controls did not differ (11.6% vs 8.6%, respectively; P = .46) using combined results of culture and PCR, independent of vaccination or behavioral risks. PWH showed altered microbiota composition (ie, β-diversity; NP: P = .0028, OP: P = .0098), decreased α-diversity (NP: P = .024, OP: P = .0045), and differences in the relative abundance of multiple bacterial taxa. Spn colonization was associated with altered β-diversity in the nasopharynx (P = .011) but not oropharynx (P = .21). Conclusions Despite widespread conjugate vaccine and antiretroviral use, frequencies of Spn colonization among PWH and controls are currently consistent with those reported in the preconjugate era. The persistently increased risk of pneumococcal disease despite antiretroviral therapy may relate to behavioral and immunologic variables other than colonization.