This study aimed to examine the reproducibility and validity of a new food frequency questionnaire (FFQ) used in a birth cohort study to estimate the usual consumption frequencies of foods during pregnancy. The reference measure was the average of three inconsecutive 24 h diet recalls (24 HR) administrated between two FFQs, and the reproducibility was measured by repeating the first FFQ (FFQ1) approximately eight weeks later (FFQ2). A total of 210 pregnant women from the Born in Guangzhou Cohort Study (BIGCS) with full data were included in the analysis. The Spearman’s correlation coefficients of FFQ1 and FFQ2 ranged from 0.33 to 0.71. The intraclass correlation coefficients of the two FFQs ranged from 0.22 to 0.71. The Spearman’s correlation coefficients of the 24 HR and FFQ2 ranged from 0.23 to 0.62. Cross-classification analysis showed 65.1% of participants were classified into same and contiguous quintiles, while only 3.2% were misclassified into the distant quintiles. Bland-Altman methods showed good agreement for most food groups across the range of frequencies between FFQ1 and FFQ2. Our findings indicated that the reproducibility and validity of the FFQ used in BIGCS for assessing the usual consumption frequencies of foods during pregnancy were acceptable.