An ultrahigh-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the profiling of six folate species in potatoes. The calibration curves cover a wide, linear range (the lower and upper limits of quantitation range between 0.22-0.24 and 216.07-242.28 μg/100 g of fresh weight), allowing sensitive determination in small amounts of potato flesh. With a single exception, the acceptance criteria for intra- and interday precision and accuracy were met: for all quality controls, the percent relative standard deviation and the percent bias were lower than 15% (or 20% at the lower limit of quantitation). Application of the method on tubers at different stages of maturation demonstrated the large variability within a single variety: the folate content and polyglutamylation rate varied between 10.35 and 24.01 μg/100 g of fresh weight and between 4.96% and 60.49%, respectively. Additionally, the two-dimensional folate profiling of mature tubers demonstrated an increase in folate from center to peel, combined with a stable species distribution and polyglutamylation rate.