Heat shock proteins (Hsps) are key components contribute to cellular homeostasis under stressful conditions. They function as molecular chaperone involved in protein folding, plant development, signal transduction and protecting plants against several stresses. In this study, we developed transgenic alfalfa by Agrobacterium-mediated genetic transformation, and integration and expression of MsHsp23was confirmed by polymerase chain reaction (PCR), Southern and Northern blot analyses. Transgenic (Tg1, Tg3, Tg4 andTg7) and non-transgenic (NT) alfalfa plants were exposed to heat (42 °C)treatment for 24 h. Compared to NT control plants, Tg plants showed less wiltingthat maintained leaves color compared to NT plants. The expression of MsHsp23gene and the ascorbate peroxidase (APX) levels were higher in MsHsp23alfalfa lines relative to NT plants. The result indicates the chaperone activity of MsHsp23genethat allows for the induction of APX in transgenic alfalfa. We conclude that high level of MsHsp23withAPX activity enhanced tolerance to heat stress in transgenic alfalfa plants.