High-performance thin-layer chromatography (HPTLC) combined with image analysis and pattern recognition methods were used for fingerprinting of phenolic compounds present in seedlings of two maize genotypes ZP 434 (new generation hybrid, drought tolerant) and ZP 704 (older generation hybrid, drought sensitive) treated with different concentrations of 24-epibrassinolide. This is the first report of TLC chromatographic profile of phenolics’ mixtures in maize seed extracts influenced by brassinosteroid phytohormones. Nine samples of shoot of seedlings for the whole concentration range of phytohormones (5.2 × 10−7-5.2 × 10−15 M), one sample of root of seedlings treated with 5.2 × 10−15 M 24-epibrassinolide, and the control samples of nontreated seedlings, for both genotypes, were analyzed. Phenolic profiles of root extracts indicate the absence of more polar compounds such as phenolic acids and glycosides present in shoot of seedlings. Also, hormones applied in higher concentrations have an inhibiting effect on the content of phenolics in ZP 434. Application of chemometric methods enables characterization of particular genotype of maize according to its phenolic profile.