Nutritional deficiencies of folate cause neural tube defects and several other diseases. The tri-enzyme method and microbiological assays were used to investigate folate variation in 78 rice germplasms. The effects of storage and cooking on folate content were also analyzed. Folate contents of brown rice varied substantially from 13.3 to 111.4 μg/100 g, whereas milled rice varied from 10.3 to 77.7 μg/100 g. Four cultivars from South China with high folate levels were identified in both sub-species. The average folate losses caused by storage and cooking were 23% and 48.3%, respectively. The highest folate content in cv. Laoshuya when cooked was 26.3 μg/100 g, about 25% of the recommended dietary intake (400 μg/day), assuming a daily per-capita consumption of 400 g/day cooked rice. The results suggested that it is potentially possible to screen for higher folate content varieties in germplasm and in breeding. It is also essential to develop new processing methods for maintaining higher folate contents in cooked rice.