Imaging fluorometry was applied to investigate the tolerance of Japanese honeysuckle (Lonicera japonica Thunb.) of different ploidy levels to heat stress. Seedlings of L. japonica, the diploid cv. Damaohua and the tetraploid cv. Jiufengyihao, were exposed to heat stress of 42 ºC for 6 h and a recovery for 10 h. Heat stress significantly decreased maximum photochemical efficiency, electron transport rate, effective quantum yield of photosystem 2, and photochemical quenching of both cultivars, but it decreased the non-photochemical quenching (NPQ) only in the tetraploid. Heat stress increased the content of total soluble sugars, proline, and malondialdehyde in both cultivars while it increased NPQ only in the diploid. Our findings suggest that the tetraploid showed to be more resistance to heat stress than the diploid of L. japonica, which was indicated by different chlorophyll fluorescence imaging techniques and metabolic changes. Moreover, the degree of recovery in the tetraploid was higher than that of the diploid. The tetraploid also possessed thicker epidermis (both upper and lower) and palisade tissue as well as denser pubescence.