Background Choline and betaine status have previously been associated with symptoms of depression. However, the relation of maternal plasma choline and betaine concentrations in pregnancy to peripartum maternal mood is unknown. Methods Maternal plasma choline and betaine concentrations (μmol/L) were measured at 26–28 weeks gestation in the Growing Up in Singapore Toward healthy Outcomes (GUSTO) mother–offspring cohort. Participants completed the State‐Trait Anxiety Inventory (STAI) and Edinburgh Postnatal Depression Scale (EDPS) at 26–28 weeks gestation (n = 949) and at 3 months postnatal (n = 689): higher scores are indicative of more symptoms of anxiety and depression. Multivariate linear regression models were used to estimate the association of choline and betaine with ante‐ and postnatal mental well‐being adjusting for covariates. Results Mean (SD) antenatal plasma choline and betaine concentrations were 9.2 μmol/L (1.6) and 13.1 μmol/L (2.7), respectively. Plasma choline concentrations were positively associated with antenatal depressive (β = .24 EPDS score [95% CI: 0.05–0.43] per μmol/L] and anxiety symptoms (β = .46 STAI‐state score [95% CI: 0.03–0.88] per μmol/L) adjusting for covariates. Plasma betaine concentrations were not associated with antenatal depression or anxiety symptoms. No associations were observed between pregnancy choline or betaine and postnatal mental well‐being. Conclusion This study suggests that higher maternal plasma choline status during pregnancy is associated with more symptoms of antenatal depression and anxiety, whereas plasma betaine concentrations showed no associations. No associations were observed for postnatal mental well‐being. Prospective studies are required to replicate these findings and further examine the direction of causality and possible biological mechanisms.