Soybean is a rich source of folates. We optimised the extraction and detection of folates from soybean seeds by HPLC-MSMS and analysed the folate content and composition of 1074 accessions. Total folate content ranged from 64.51 to 691.24 μg100 g fresh weight, with 10-fold variation, and 60 elite accessions with over 400 μg100 g of total folate were identified. The most abundant component was 5-CHO-H4folate, which accounted for an average of 60% of total folate content. Seed-coat colour, seed weight, ecoregion, and accession type significantly affected soybean folate content. Furthermore, 5-CH3-H4folate correlated positively with seed protein (r =0.24***) and negatively with oil (r = -0.26***). The geographical distribution of folate according to accession origin revealed that accessions from Northeast China contain higher amounts of total folate and 5-CHO-H4folate. This study provides comprehensive and novel insights into the folate profile of soybean, which will benefit soybean breeding for folate enhancement.