The consumption of beetroot, a food rich in nitrate, has been recommended to enhance blood perfusion, restore endothelial function, and improve exercise performance. These properties may be explained by the possible effect of nitrate present in beetroot in stimulating the endogenous synthesis of nitric oxide, a potent vasodilator. However, there is limited evidence giving detailed information about how nitrate contents from beetroot used in studies have been analyzed. The purpose of this study was to evaluate nitrate and nitrite contents of beetroot from different regions of Brazil and from the USA. Nitrate and nitrite contents were quantified by using a reversed-phase high-performance liquid chromatography system with fluorescence detection. Beetroots from the USA showed the highest nitrate (31.2 ± 0.010 mmol.L−1) and nitrite (0.45 ± 0.005 mmol.L−1) contents when compared to beetroots from Brazil. Rio de Janeiro was the region that showed the highest nitrate content (17.1 ± 0.020 mmol.L−1), while Rio Grande do Norte presented the highest nitrite content (0.13 ± 0.010 mmol.L−1). The reversed-phase high-performance liquid chromatography-fluorescence method may be applied for the quantification of nitrate and nitrite contents in beetroot samples. The results of the present study demonstrate that beetroots may vary in their nitrate and nitrite contents. This data represents a useful tool to encourage researchers to analyze the nitrate and nitrite contents in beetroots utilized in interventional clinical studies.