The 5-methyltetrahydrofolate( 5-MTHF) content in different fruit tissues of nine citrus varieties were determined by Reversed-phase High Performance Liquid Fluorescence Detection method( RP-HPLC-FLD). Results indicated that 5-MTHF content in the pulp of sweet oranges( Citrus Sinensis) was the highest,especially that in the Hamlin pulp was up to 29. 28 μg /100g; followed by pummelo varieties( C. grandis),the content of 5-MTHF in Shatian pummelo pulp was 23. 45μg /100g,liangping pummelo 15. 01μg /100g; Mandarin fruits had lower content of5-MTHF,Satsuma Mandarin pulp had 15. 58μg /100g,Taiwan Ponkan only 8. 92μg /100g; the lowest content of 5-MTHF was in Eureka Lemon pulp,only 4. 1μg /100g. In general,the content of 5-MTHF in different fruit tissues from high to low was: seed > pulp > segment wall > peel.