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This study aimed optimizes the inductively coupled plasma–atomic emission spectrometry (ICP-AES) conditions for the determination of macro and microelements in the flowers and leaves of Trifolium species. After the optimization of instrumental parameters, plasma robustness was achieved under the following conditions: RF power 1350 W, nebulizer flow rate 0.5 L min−1, coolant gas flow rate 12 L min−1, auxiliary gas flow rate 0.5 L min−1, flush pump rate 100 rpm, analysis pump rate 50 rpm, sample flow 0.5 mL min−1, and sample uptake delay 30 s. The limits of detection (LOD) and limits of quantification (LOQs) were lower than literature values. The most abundant microelement in leaves is Ca, followed by K, Mg, P, and Na, and in flowers was K, followed by Ca, P, Mg, and Na. The average contents of microelements decreased in the orders S > Fe > Si > Al > Zn > Mn > Cu > Ni > Co > Pb > Cr > As > Cd for flowers and S > Fe > Si > Mn > Al > Zn > Cu > Ni > Co > Pb > Cr > As > Cd for leaves. Chemometrics were used for grouping samples based upon the elemental contents. [ABSTRACT FROM AUTHOR]