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Vaginal dysbiosis has been shown to increase the risk of some adverse birth outcomes. HIV infection may be associated with shifts in the vaginal microbiome. We characterized microbial communities in vaginal swabs collected between 16–20 gestational weeks in the Zambian Preterm Birth Prevention Study to investigate whether HIV and its treatment alter the microbiome in pregnancy. We quantified relative abundance and diversity of bacterial taxa by whole-genome shotgun sequencing and identified community state types (CST) by hierarchical clustering. Associations between exposures—HIV serostatus (HIV+ vs HIV-) and preconceptional ART (ART+ vs ART-)—and microbiome characteristics were tested with rank-sum, and by linear and logistic regression, accounting for sampling by inverse-probability weighting. Of 261 vaginal swabs, 256 (98%) had evaluable sequences; 98 (38%) were from HIV+ participants, 55 (56%) of whom had preconceptional ART exposure. Major CSTs were dominated by: L. crispatus (CST 1; 17%), L.] iners (CST 3; 32%), Gardnerella vaginalis (CST 4-I; 37%), G. vaginalis & Atopobium vaginae (CST 4-II; 5%), and other mixed anaerobes (CST 4-III; 9%). G. vaginalis was present in 95%; mean relative abundance was higher in HIV+ (0.46±0.29) compared to HIV- participants (0.35±0.33; rank-sum p = .01). Shannon diversity was higher in HIV+/ART+ (coeff 0.17; 95%CI (0.01,0.33), p = .04) and HIV+/ART- (coeff 0.37; 95%CI (0.19,0.55), p < .001) participants compared to HIV-. Anaerobe-dominant CSTs were more prevalent in HIV+/ART+ (63%, AOR 3.11; 95%CI: 1.48,6.55, p = .003) and HIV+/ART- (85%, AOR 7.59; 95%CI (2.80,20.6), p < .001) compared to HIV- (45%). Restricting the comparison to 111 women in either CST 3 (L. iners dominance) or CST 1 (L. crispatus dominance), CST 3 frequency was similar in HIV- (63%) and HIV+/ART- participants (67%, AOR 1.31; 95%CI: (0.25,6.90), p = .7), but higher in HIV+/ART+ (89%, AOR 6.44; 95%CI: (1.12,37.0), p = .04). Pregnant women in Zambia, particularly those with HIV, had diverse anaerobe-dominant vaginal microbiota. [ABSTRACT FROM AUTHOR]