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Heat shock transcription factors (HSF) are divided into classes A, B and C. Class A transcription factors are generally recognized as transcriptional activators, while functional characterization of class B and C heat shock transcription factors have not been fully developed in most plant species. We isolated and characterized a novel HSF transcription factor gene, <i>TrHSFB2a</i> (a class B HSF) gene, from the drought stress-sensitive forage crop species, white clover (<i>Trifolium repens</i>). <i>TrHSFB2a</i> was highly homologous to <i>MtHSFB2b</i>, <i>CarHSFB2a</i>, <i>AtHSFB2b</i> and <i>AtHSFB2a.</i> The expression of <i>TrHSFB2a</i> was strongly induced by drought (<i>PEG6000</i> 15% <i>w</i>/<i>v</i>), high temperature (35 °C) and salt stresses (200 mM L⁻¹ NaCl) in white clover, while subcellular localization analysis showed that it is a nuclear protein. Overexpression of the white clover gene <i>TrHSFB2a</i> in <i>Arabidopsis</i> significantly reduced fresh and dry weight, relative water contents (RWC), maximum photosynthesis efficiency (Fv/Fm) and performance index on the absorption basis (PI_ABS), while it promoted leaf senescence, relative electrical conductivity (REC) and the contents of malondialdehyde (MDA) compared to a wild type under drought, heat and salt stress conditions of <i>Arabidopsis</i> plants. The silencing of its native homolog (<i>AtHSFB2a</i>) by RNA interference in <i>Arabidopsis thaliana</i> showed opposite trends by significantly increasing fresh and dry weights, RWC, maximum photosynthesis efficiency (Fv/Fm) and performance index on the absorption basis (PI_ABS) and reducing REC and MDA contents under drought, heat and salt stress conditions compared to wild type <i>Arabidopsis</i> plants. These phenotypic and physiological indicators suggested that the <i>TrHSFB2a</i> of white clover functions as a negative regulator of heat, salt and drought tolerance. The bioinformatics analysis showed that <i>TrHSFB2a</i> contained the core B3 repression domain (BRD) that has been reported as a repressor activator domain in other plant species that might repress the activation of the heat shock-inducible genes required in the stress tolerance process in plants. The present study explores one of the potential causes of drought and heat sensitivity in white clover that can be overcome to some extent by silencing the <i>TrHSFB2a</i> gene in white clover.