The bio-enhancement breeding for higher folate in food crops is a potential effective way to improve folate deficiency in humans. It is of great significance to study the genetic effects of folate traits in waxy maize F0 kernels. Two components 5-methyltetrahydrofolate (5-M-thf) and 5-formyltetrahydrofolate (5-F-thf) are the main forms of natural folate. In order to the content and variants of folate in F0 kernels, twenty crosses were generated according to Griffing diallel cross using 5 waxy corn inbred lines, followed by the evaluation of the 5-M-thf and 5-F-thf contents of fresh F0 kernels in this study. The results showed that the content of 5-M-thf was higher than that of 5-F-thf in all crosses, and the content of 5-M-thf in ZN3/J6 was highest, which was 298.40ug/100g DW; the content of 5-F-thf in BN2/ZN3 was the highest, which was 59.40ug/100g DW. The F0 heterosis was observed in the 5-M-thf and 5-F-thf. The 5-F-thf showed positive heterosis in all crosses, while the 5-M-thf showed variants on heterosis in different crosses. Further calculation of the heritability and maternal effect of folate traits showed that the maternal effect of 5-M-thf (0.28) was higher than that of 5-F-thf, while the heritability of 5-M-thf (0.81) was higher than that of 5-F-thf. In conclusion, this study revealed the F0 heterosis and maternal effect of folate trait in waxy maize kernels, and the different performance on forms of folate in maize kernels. The inbred lines such as BN2 and ZN3, which were detected with high folate content, can be applied as female parent to obtain high folate content in maize F0 kernels.