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OBJECTIVE An increase in the number of women who have fear of birth [FOB] has been reported globally; yet, how these women are identified varies. This study aimed to identify the most effective way of measuring FOB in clinical practice. DESIGN This paper reports on a prospective cohort study; a core element of an explanatory mixed-methods study. This element explored the appropriateness of measures of anxiety (biomarkers and validated questionnaires) and observed any relationship between anxiety levels and clinical outcomes. PARTICIPANTS A purposive sampling strategy was used. One hundred and forty-eight primigravida, during the 1st trimester, in two tertiary maternity units in England were included. METHODS Demographic and baseline data were collected from participants in the first trimester of pregnancy along with FOB scores, and a saliva sample to measure cortisol level. In the third trimester, a second FOBS score, and saliva sample were collected, and the Personal Health Questionnaire-9 (PHQ-9) and General Anxiety Disorder-7 (GAD-7) were administered to measure depression and anxiety respectively. FINDINGS The FOBS was completed by 148 women in the first trimester and 80 in the third. Using a cut-off of 54, 30/148 (20%) women had a FOB in the first trimester; 21/80 (26%) had a FOB in the third trimester, 15 (19%) of whom also had a FOB in the first. Compared with the first trimester, 51/80 women showed an increase in FOBS score, with 14 scores increasing above and 8 scores decreasing below the cut-off of 54. FOBS scores were not correlated with salivary cortisol in either trimester (first trimester Spearman's ρ=0.08, p = 0.354, n = 144; third trimester ρ=0.12, p = 0.309, n = 71) but they were correlated with PHQ-9 and GAD-7 scores in the third trimester (PHQ-9 ρ=0.53, p = 0.010, n = 23; GAD-7 ρ=0.45, p = 0.033, n = 23) although not sufficiently high enough to demonstrate convergent validity against those measures of depression and anxiety. They were also associated with a previous history of depression but only in the first trimester (p = 0.011). FOBS scores showed considerable variability and a high measurement error, indicating a need for further refinement and psychometric testing. CONCLUSION The FOBS is a potentially effective way of measuring FOB in clinical practice and research, but it requires refining. Scores are not related to salivary cortisol levels but are correlated with validated scores for anxiety and depression. An enhanced version of the FOBS could be used in clinical practice to measure FOB.