Omega-6 (n-6) and omega-3 (n-3) polyunsaturated fatty acids (PUFAs) are vital for fetal metabolic programming and immunomodulation. Higher n-6:n-3 ratios, reflecting a proinflammatory eicosanoid profile, are associated with adverse perinatal outcomes. Limited data exist, however, on n-6 and n-3 PUFAs specifically in the context of HIV and pregnancy. Our objective was to assess HIV clinical factors associated with PUFA signatures in pregnant persons with HIV (PWH). In this observational cohort, third trimester plasma PUFA concentrations (six n-6 PUFAs, four n-3 PUFAs) were measured, each as a percent of total fatty acid content, via esterification and gas chromatography in pregnant PWH enrolled from 2009 to 2011 in the Nutrition substudy of the Pediatric HIV/AIDS Cohort Study. PUFA ratios (n-6:n-3) were calculated. Exposures assessed were first/second trimester CD4 count (<200 vs. >200 cells/mm3), HIV RNA viral load (VL) (VL >400 vs. <400 copies/mL), and protease inhibitor (PI) versus non-PI antiretroviral therapy (ART). Linear regression models using generalized estimating equations were fit to assess mean differences and their 95% confidence intervals (CIs) in n-6:n-3 by each exposure, adjusted for potential confounders. Of 264 eligible pregnant PWH, the median age was 27 years, 12% had CD4 counts <200 cells/mm3, and 56% had VL ≥400 copies/mL in the first/second trimesters. PUFA concentrations and ratios were similar by CD4 count and PI exposure. n-3 concentrations were lower in PWH with VL ≥400 versus <400 copies/mL (median 2.8% vs. 3.0%, p < .01, respectively); no differences were observed for n-6 concentrations by VL. In models adjusted for age, education, tobacco use, body mass index, and PI-based ART, n-6:n-3 was higher in those with VL ≥400 copies/mL (mean difference: 1.6; 95% CI: 0.79-2.48, p = .0001). Therefore, PUFA signatures in viremic pregnant PWH reflect a proinflammatory eicosanoid milieu. Future studies should evaluate associations of proinflammatory PUFA signatures with adverse perinatal outcomes in PWH.