Emerging but inconsistent evidence suggests that glyphosate (GLY)-based herbicides (GBHs) are more toxic than the active ingredient, GLY alone. Polyoxyethylene tallow amines (POEAs) are surfactants most widely used in GBH formulations, making up to 5–15 % by weight. However, neither POEA exposure nor their toxicity has been well studied in the general population. This study leverages second trimester urine samples collected from 86 pregnant participants of a multi-center, US-based pregnancy cohort. We measured three POEA homologs, i.e., C16s(EO)2, C18u(EO)2, and C18s(EO)2, using an ultrahigh performance liquid chromatography-tandem with mass spectrometry. These homologs were detected in 79 %, 97 %, and 59 % of the samples (LOD: 0.1 ng/mL) at a mean concentration of 1.87, 1.88, and 1.65 ng/mL, respectively. While these homologs were highly correlated with each other (rho >0.7), their correlations with GLY and its breakdown product, aminomethylphosphonic acid (AMPA), were moderate (rho <0.4). Compared to other races, the POEA level in urine was the lowest in White participants (p = 0.047). We observed an indication that a higher POEA level was associated with increased ano-genital distance (AGD), a marker of gestational androgen milieu, in male infants only (β = 2.46, p = 0.057); such relationship remained unchanged after adjusting for GLY (β = 2.45, p = 0.055) or AMPA (β = 2.46, p = 0.058). Despite the small sample size, our study provides the first population data on POEA exposure which demonstrates widespread exposure in the general population. Suggestive associations between urinary POEA and AGD in male newborns implicate potential endocrine disrupting property of POEA, which warrant further investigation in larger studies.