Introduction. Anxiety, mood- and stress-related behaviors are regulated by sex hormones in pregnant and non-pregnant women. Very scarce information exists about the role of sex steroids in pregnant women displaying high levels of anxiety. Objective. To determine sex hormones serum levels in pregnant women exhibiting high levels of anxiety symptoms. Method. The Hamilton Anxiety Rating Scale (HARS/ HAM-A) was used to assess the intensity of anxiety symptoms in third-trimester pregnant women. Two groups were included in the study, pregnant women exhibiting severe anxiety (ANX; HARS scores ≥ 25; n = 101) and healthy control subjects (CTRL; n = 40) displaying lower scores for anxiety (HARS scores ≤ 7). Estradiol (E2), progesterone (P4), and testosterone (T) serum levels were measured using a standard chemiluminescent immunoassay. Bivariate and partial correlations were performed to detect significant associations between groups, clinical measures, biochemical data, and HARS scores. Results. The anxiety group (ANX) showed an increase in E2 and T serum levels (p () .001) compared to CTRL. Conversely, significantly lower P4 levels were found in the symptomatic group (p () .001) as compared to the CTRL hormone values. The P4:E2 index was significantly reduced in pregnant women with high levels of anxiety (p () .001). Negative correlations between anxiety (HARS) scores, P4 serum levels (p = .02), and P4:E2 ratio (p = .04) were found in the symptomatic group. Conversely, T serum levels displayed a positive association (p = .001) with high levels of anxiety symptoms in the same group, after adjusting our data by clinical confounders. Discussion and conclusion. Serum levels of sex-steroid hormones are altered in pregnant women exhibiting severe anxiety.