The purpose of this study was to determine the difference in vitamin B6 content according to the varieties, regions, and harvest times of vegetables and fruits in Korea using high performance liquid chromatography. We verified the accuracy of the analytical method with standard reference material 1849a and achieved reliability for internal analysis quality control with a mixture of cereal, whole wheat, and flour. As a result of the analysis, vitamin B6 contents (μg/100 g) were 6.9~86.5 in peppers, 5.1~17.2 in paprika, 4.4~5.0 in strawberries, 4.0~52.9 in tomatoes, 7.7~7.8 in Chinese cabbage, 17.3~23.3 in radishes, 13.4~37.6 in apples, 2.3~12.7 in peaches, and 3.7~12.7 in grapes. In general, the difference in vitamin B6 content showed by varieties, harvest times, and regions. Peppers showed the most difference as 79.6 μg/100 g among the varieties, and apples showed a difference as 22.8 μg/100 g by regions. According to the harvest times in grape ‘Mihwang’, there was a difference of 7.5 μg/100 g. We will collect an amount of nutritional data on various food materials, and continue to build a reliable and integrated nutritional database. And then the database will be used in the 10th revision of the Korean Food Composition Table.