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Background: The dietary nutritional status of the lactating mothers is related to maternal health and has a significant impact on the growth and development of infants through the secretion of breast milk. The food frequency questionnaire (FFQ) is the most cost-effective dietary assessment method that can help obtain information on the usual dietary pattern of participants. Until now, the FFQs have been used for different populations in China, but there are few FFQs available for the lactating mothers. We aimed to develop a semi-quantitative, 156-item FFQ for the Chinese lactating mothers, and evaluate its reproducibility and relative validity., Methods: A total of 112 lactating mothers completed two FFQs and one 3-d dietary record (3DR). The first FFQ (FFQ1) was conducted during postpartum at 60-65 days and the second FFQ (FFQ2) during subsequent follow-up at 5 weeks. The 3DR was completed with portion sizes assessed using photographs taken by the respondent before and after eating (instant photography) 1 week after FFQ1., Results: For reproducibility, the Spearman's correlation coefficients for food ranged from 0.34 to 0.68, and for nutrients from 0.25 to 0.61. Meanwhile, the intra-class correlation coefficients for food ranged from 0.48 to 0.87, and for nutrients from 0.27 to 0.70. For relative validity, the Spearman's correlation coefficients for food ranged from 0.32 to 0.56, and for nutrients from 0.23 to 0.72. The energy-adjusted coefficients for food ranged from 0.26 to 0.55, and for nutrients from 0.22 to 0.47. Moreover, the de-attenuation coefficients for food ranged from 0.34 to 0.67, and for nutrients from 0.28 to 0.77. The Bland-Altman plots also showed reasonably acceptable agreement between the two methods., Conclusions: This FFQ is a reasonably reproducible and a relative valid tool for assessing dietary intake of the Chinese lactating mothers.