Hemp (Cannabis sativa L.) seedlings do not contain cannabinoids, which are the species' typical active compounds, but synthesize flavonoids, including the anti-inflammatory compounds cannflavin A and B. The aim of this study was to analyse the content of flavonoids in different commercial hemp cultivars, Fibrol, Finola, Epsilon 68, Secuieni Jubileum, Fedora, Henola, Uso 31, Earlina 8FC, Kompoliti, and Lipko, using the HPLC-MS/MS method. The plants were grown in pots at temperature of 25°C (±2°C) under a 16-hour photoperiod with 80–100 μmol m⁻² s⁻¹ fluorescent daylight. Fifteen days after germination, the aerial parts were collected, weighed, and dried. The catechin, vitexin, cannflavins (A and B), quercetin, isovitexin, apigenin, orientin, naringenin, luteolin, and myricetin content was determined in the isopropanol extracts. The chromatographic analysis was performed using a Shimadzu 8040 LC-MS chromatograph and Zorbax SB-C18 column (3.0 mm x 150 mm 3.5 μm). Mobile phase A consisted of acetonitrile and formic acid solution in water 0.1% (V/V), both HPLC grade. Mobile phase B consisted of water and formic acid at a concentration of 0.1% (V/V). The analysis was performed in negative ion charge, using multiple reaction monitoring (MRM) for qualitative and quantitative analyses. The analysis was performed in triplicate (3 n). Statistical significance was determined applying Duncan’s post hoc (p value of 0.05). Significant variation in flavonoid content was found. The Kompolti cultivar had the most diverse flavonoid profile (cannflavin A and B, isovitexin, vitexin, and apigenin). The highest concentrations of cannflavin A were recorded for the Epsilon 68 (18.3 ± 1.1 µg/g) and Fibrol (18.2 ± 4.3 µg/g) cultivars. The content of cannflavin B was much lower and ranged from 1.2 to 2.5 µg/g. Genotypes such as Kompolti, Epsilon 68, and Fibrol are potential sources of valuable flavonoids and can be used as parental material in further breeding work.